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Abstract:

The invention relates to materials and methods of conjugating a water
soluble polymer to an oxidized carbohydrate moiety of a therapeutic
protein comprising contacting the oxidized carbohydrate moiety with an
activated water soluble polymer under conditions that allow conjugation.
More specifically, the present invention relates to the aforementioned
materials and methods wherein the water soluble polymer contains an
active aminooxy group and wherein an oxime or hydrazone linkage is formed
between the oxidized carbohydrate moiety and the active aminooxy group on
the water soluble polymer, and wherein the conjugation is carried out in
the presence of a nucleophilic catalyst.

3. The method according to claim 2 wherein a solution comprising an
initial concentration of the therapeutic protein between about 0.3 mg/ml
and about 3.0 mg/ml is adjusted to a pH value between about 5.0 and about
8.0 prior to contacting with the activated water soluble polymer.

4. The method of claim 3 wherein the initial concentration of the
therapeutic protein is about 1.0 mg/ml and the pH is about 6.0.

5. The method of claim 2 wherein the therapeutic protein is contacted by
a desired excess concentration of activated water soluble polymer,
wherein the excess concentration is between about 1-molar and about
300-molar excess.

6. The method of claim 5 wherein the excess concentration is about
50-fold molar excess.

7. The method of claim 5 wherein the therapeutic protein is incubated
with the activated water soluble polymer under conditions comprising a
time period between about 0.5 hours and about 24 hours; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence or
absence of light; and with or without stirring.

8. The method according to claim 7 wherein the conditions comprise a time
period of about 120 minutes, a temperature of about 22.degree. C., the
absence of light; and with stirring.

9. The method according to claim 2 wherein the nucleophilic catalyst is
added in an amount to result in a final concentration between about 1.0
mM and about 50 mM nucleophilic catalyst, under conditions comprising a
time period between about 0.1 minutes and about 30 minutes; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence or
absence of light; and with or without stirring.

10. The method of claim 9 wherein the final concentration of the
nucleophilic catalyst is about 10 mM, and the conditions comprise a time
period of up to about 15 minutes, a temperature of about 22.degree. C.,
the absence of light; and with stirring.

11. The method according to claim 2 wherein the oxidizing agent is added
in an amount to result in a final concentration between about 50 μM
and about 1000 μM oxidizing agent, under conditions comprising a time
period between about 0.1 minutes and 120 minutes; a temperature between
about 2.degree. C. and about 37.degree. C.; in the presence or absence of
light; and with or without stirring.

12. The method of claim 11 wherein the final concentration of oxidizing
agent is about 400 μM, and the conditions comprise a time period of
about 10 minutes, a temperature of about 22.degree. C., the absence of
light and with stirring.

13. The method of claim 2 wherein the conjugating the water soluble
polymer to the oxidized carbohydrate moiety of the therapeutic protein is
stopped by the addition of a quenching agent selected from the group
consisting of L-cysteine, methionine, glutathione, glycerol, sodium meta
bisulfate (Na2S2O5), tryptophane, tyrosine, histidine or
derivatives thereof, kresol, imidazol, and combinations thereof; wherein
the quenching agent is added in an amount to result in a final
concentration between about 1 mM and about 100 mM quenching agent, under
conditions comprising a time period between about 5 minutes and about 120
minutes; a temperature between about 2.degree. C. and about 37.degree.
C.; in the presence or absence of light; and with or without stirring.

14. The method of claim 13 wherein the quenching agent is L-cysteine.

15. The method of claim 14 wherein the L-cysteine is added to result in a
final concentration of about 10 mM and the conditions comprise a time
period of about 60 minutes, a temperature of about 22.degree. C., the
absence of light and with stirring.

16. The method of claim 2 comprising: a) a first step comprising
adjusting the pH value of a solution comprising the therapeutic protein
to a pH value between about 5.0 and about 8.0, wherein the therapeutic
protein concentration is between about 0.3 mg/ml and about 3.0 mg/ml; b)
a second step comprising oxidizing one or more carbohydrates on the
therapeutic protein, wherein the oxidizing agent is added to the solution
in the first step to result in a final concentration between about 50
μM and about 1000 μM, under conditions comprising a time period
between about 0.1 minutes and about 120 minutes; a temperature between
about 2.degree. C. and about 37.degree. C.; in the presence or absence of
light, and with or without stirring; c) a third step comprising
contacting the therapeutic protein with a desired excess concentration of
activated water soluble polymer, wherein the excess concentration is
between about 1-molar excess and about 300-molar excess, under conditions
comprising a time period between about 0.5 hours and about 24 hours, a
temperature between about 2.degree. C. and about 37.degree. C.; in the
presence or absence of light; and with or without stirring; d) a fourth
step comprising adding a nucleophilic catalyst to the solution of the
third step, wherein the nucleophilic catalyst is added to result in a
final concentration between about 1 mM and about 50 mM, under conditions
comprising a time period between about 0.1 minutes and about 30 minutes;
a temperature between about 2.degree. C. and about 37.degree. C.; in the
presence or absence of light, and with or without stirring; e) a fifth
step wherein the therapeutic protein is incubated with the activated
water soluble polymer and nucleophilic catalyst under conditions that
allow conjugation of the activated water-soluble polymer to one or more
oxidized carbohydrates on the therapeutic protein, said conditions
comprising a time period between about 0.5 hours and about 24 hours, a
temperature between about 2.degree. C. and about 37.degree. C.; in the
presence or absence of light, and with or without stirring; and f) a
sixth step wherein the conjugating the water soluble polymer to the one
or more oxidized carbohydrates of the therapeutic protein in the fifth
step is stopped by the addition of a quenching agent selected from the
group consisting of L-cysteine, methionine, glutathione, glycerol,
Na2S2O5 (sodium meta bisulfite), tryptophane, tyrosine,
histidine or derivatives thereof, kresol, imidazol, and combinations
thereof; wherein the quenching agent is added to result in a final
concentration of about 1 mM and about 100 mM, under conditions comprising
a time period between about 5 minutes and about 120 minutes; a
temperature between about 2.degree. C. and about 37.degree. C.; in the
presence or absence of light, and with or without stirring.

17. The method of claim 16 wherein the initial concentration of the
therapeutic protein in the first step is about 1 mg/ml and the pH is
about 6.0; wherein the final concentration of oxidizing agent in the
second step is about 400 μM, and the conditions in the fifth step
comprise a time period of about 10 minutes, a temperature of about
22.degree. C., the absence of light and with stirring; wherein the excess
concentration in the third step is about 50 molar excess; wherein the
conditions in the third step comprise a time period of about 15 minutes,
a temperature of about 22.degree. C., the absence of light and with
stirring; wherein the final concentration of the nucleophilic catalyst in
the fourth step is about 10 mM, and the conditions in the fourth step
comprise a time period of about 15 minutes, a temperature of about
22.degree. C., the absence of light and with stirring; wherein the
conditions of incubating the therapeutic protein with the activated water
soluble polymer and nucleophilic catalyst in the fifth step comprise a
time period of about 2 hours; a temperature of about 22.degree. C.; the
absence of light; and with stirring; and wherein the quenching agent in
the sixth step is L-cysteine; and wherein the L-cysteine is added to
result in a final concentration of about 10 mM and the conditions in the
sixth step comprise a time period of about 60 minutes, a temperature of
about 22.degree. C., the absence of light and with stirring.

18. The method according to claim 2 wherein the water soluble polymer is
PSA.

19. The method according to claim 2 wherein the water soluble polymer is
PEG.

20. The method according to claim 2 wherein the water soluble polymer is
HES.

21. The method according to claim 2 wherein the water soluble polymer is
HAS.

22. The method according to claim 18 wherein the PSA is comprised of
about 10-300 sialic acid units.

23. The method according to claim 2 wherein the therapeutic protein is
FIX.

24. The method according to claim 2 wherein the therapeutic protein is
FVIIa.

25. The method according to claim 2 wherein the therapeutic protein is
FVIII.

26. The method according to claim 2 wherein the oxidizing agent is sodium
periodate (NaIO4).

27. The method according to claim 25 wherein the oxidized carbohydrate
moiety of the therapeutic protein is located in the activation peptide of
the blood coagulation protein.

28. The method according to claim 18 wherein the PSA is prepared by
reacting an activated aminooxy linker with oxidized PSA; wherein the
aminooxy linker is selected from the group consisting of: a) a
3-oxa-pentane-1,5-dioxyamine linker of the formula: ##STR00025## b) a
3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:
##STR00026## and c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine
linker of the formula: ##STR00027## wherein the PSA is oxidized by
incubation with a oxidizing agent to form a terminal aldehyde group at
the non-reducing end of the PSA.

29. The method according to claim 28 wherein the aminooxy linker is
3-oxa-pentane-1,5-dioxyamine.

30. The method according to claim 2 wherein the oxidizing agent is
NaIO.sub.4.

31. The method according to claim 2 wherein the nucleophilic catalyst is
provided at a concentration between about 1 mM and about 50 mM.

32. The method according to claim 31 wherein the nucleophilic catalyst is
m-toluidine.

33. The method according to claim 32 wherein the m-toluidine is present
in the conjugation reaction at a concentration of about 10 mM.

34. The method according to claim 2 further comprising the step of
reducing an oxime linkage in the conjugated therapeutic protein by
incubating the conjugated therapeutic protein in a buffer comprising a
reducing compound selected from the group consisting of sodium
cyanoborohydride (NaCNBH3), ascorbic acid (vitamin C) and
NaBH.sub.3.

35. The method according to claim 34 wherein the reducing compound is
sodium cyanoborohydride (NaCNBH3).

36. The method according to claim 2 further comprising the step of
purifying the conjugated therapeutic protein.

37. The method according to claim 36 wherein the conjugated therapeutic
protein is purified by a method selected from the group consisting of
chromatography, filtration and precipitation.

39. The method of claim 38 wherein an anti-chaotropic salt is used in a
chromatography loading step and in a chromatography washing step.

40. The method of claim 38 wherein the chromatography takes place in a
column.

41. The method of claim 40 wherein the column comprises a chromatography
resin selected from the group consisting of Phenyl-Sepharose FF and
Butyl-Sepharose FF.

42. The method of claim 41 wherein the resin is present in the column at
a bed height of between about 5 cm and about 20 cm.

43. The method according to claim 42 wherein the bed height is about 10
cm.

44. The method of claim 40 comprising one or more washing steps wherein
flow direction is set to up-flow and wherein the flow rate is between
about 0.2 cm/min and about 6.7 cm/min.

45. The method according to claim 44 wherein the flow rate is about 2
cm/min.

46. The method of claim 40 comprising one or more elution steps wherein
flow direction is set to down-flow and wherein the flow rate is between
about 0.1 cm/min and about 6.7 cm/min.

47. The method according to claim 46 wherein the flow rate is about 1
cm/min.

48. The method of claim 36 further comprising concentrating the
conjugated therapeutic protein by ultra-/diafiltration (UF/DF).

49. The method of claim 36 wherein the final concentration of therapeutic
protein is between about 0.5 and about 3 mg/ml.

50. The method according to claim 36 wherein the therapeutic protein
comprises between about 5 and about 11 water soluble polymer moieties.

51. The method of claim 2 wherein the conjugated therapeutic protein is
purified using chromatography; wherein an anti-chaotropic salt is used
for a loading step and for a washing step; the method comprising one or
more washing steps wherein flow direction is set to up-flow and wherein
the flow rate is between about 0.2 cm/min and about 6.7 cm/min and one or
more elution steps wherein flow direction is set to down-flow and wherein
the flow rate is between about 0.2 cm/min and about 6.7 cm/min; further
comprising concentrating the conjugated therapeutic protein by
ultra-/diafiltration (UF/DF).

52. The method of claim 51 wherein the chromatography is hydrophobic
interaction chromatography (HIC); wherein the one or more washing steps
flow rate is about 2 cm/min; and wherein the one or more elution steps
flow rate is about 1 cm/min.

53. A modified therapeutic protein produced by the method according to
claim 2.

58. The method according to claim 2 wherein the water soluble polymer
containing an active aminooxy group is prepared by a method comprising:
a) incubating a solution comprising an oxidized water-soluble polymer
with an activated aminooxy linker comprising an active aminooxy group
under conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated aminooxy
linker, said conditions comprising a time period between about 1 minute
and about 24 hours; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or without
stirring; thereby forming a water soluble polymer containing an active
aminooxy group; and b) purifying the water soluble polymer containing an
active aminooxy group by a method selected from the group consisting of
chromatography, filtration and precipitation.

59. The method according to claim 2 wherein the water soluble polymer
containing an active aminooxy group is prepared by a method comprising:
a) incubating a solution comprising an oxidized water-soluble polymer
with an activated aminooxy linker comprising an active aminooxy group
under conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated aminooxy
linker, said conditions comprising a time period between about 1 minute
and about 24 hours; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or without
stirring; thereby forming a water soluble polymer containing an active
aminooxy group; b) incubating a solution comprising the water soluble
polymer containing an active aminooxy group of step a) with a reducing
agent under conditions that allow the formation of a stable alkoxamine
linkage between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between about 1
minute and about 24 hours; a temperature between about 2.degree. C. and
about 37.degree. C.; in the presence or absence of light; and with or
without stirring; and c) purifying the water soluble polymer containing
an active aminooxy group by a method selected from the group consisting
of chromatography, filtration and precipitation.

60. The method according to claim 2 wherein the water soluble polymer
containing an active aminooxy group is prepared by a method comprising:
a) incubating a solution comprising an oxidized water-soluble polymer
with an activated aminooxy linker comprising an active aminooxy group
under conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated aminooxy
linker, said conditions comprising a time period between about 1 minute
and about 24 hours; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or without
stirring; thereby forming a water soluble polymer containing an active
aminooxy group; b) incubating a solution comprising the water soluble
polymer containing an active aminooxy group of step a) with a
nucleophilic catalyst under conditions comprising a time period between 1
minute and 24 hours; a temperature between 2.degree. C. and 37.degree.
C.; in the presence or absence of light; and with or without stirring;
and c) purifying the water soluble polymer containing an active aminooxy
group by a method selected from the group consisting of chromatography,
filtration and precipitation.

61. The method according to claim 2 wherein the water soluble polymer
containing an active aminooxy group is prepared by a method comprising:
a) incubating a solution comprising an oxidized water-soluble polymer
with an activated aminooxy linker comprising an active aminooxy group
under conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated aminooxy
linker, said conditions comprising a time period between about 1 minute
and about 24 hours; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or without
stirring; thereby forming a water soluble polymer containing an active
aminooxy group; b) incubating a solution comprising the water soluble
polymer containing an active aminooxy group of step a) with a
nucleophilic catalyst under conditions comprising a time period between 1
minute and 24 hours; a temperature between 2.degree. C. and 37.degree.
C.; in the presence or absence of light; and with or without stirring; c)
incubating a solution comprising the water soluble polymer containing an
active aminooxy group of step b) with a reducing agent under conditions
that allow the formation of a stable alkoxamine linkage between the
oxidized water-soluble polymer and the activated aminooxy linker, said
conditions comprising a time period between about 1 minute and about 24
hours; a temperature between about 2.degree. C. and about 37.degree. C.;
in the presence or absence of light; and with or without stirring; and d)
purifying the water soluble polymer containing an active aminooxy group
by a method selected from the group consisting of chromatography,
filtration and precipitation.

63. The method according to claim 62 wherein the water-soluble polymer is
PSA.

64. The method according to claim 62 wherein the oxidizing agent is
NaIO.sub.4.

65. The method according to claim 58 wherein the aminooxy linker is
selected from the group consisting of: a) a 3-oxa-pentane-1,5-dioxyamine
linker of the formula: ##STR00028## b) a
3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:
##STR00029## and c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine
linker of the formula: ##STR00030##

66. The method according to claim 59 wherein the reducing agent is
selected from the group consisting of sodium cyanoborohydride
(NaCNBH3), ascorbic acid (vitamin C) and NaBH.sub.3.

67. The method according to claim 66 wherein the reducing agent is sodium
cyanoborohydride (NaCNBH3).

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to materials and methods for
conjugating a water soluble polymer to a protein.

BACKGROUND OF THE INVENTION

[0002] The preparation of conjugates by forming a covalent linkage between
the water soluble polymer and the therapeutic protein can be carried out
by a variety of chemical methods. PEGylation of polypeptide drugs
protects them in circulation and improves their pharmacodynamic and
pharmacokinetic profiles (Harris and Chess, Nat Rev Drug Discov. 2003;
2:214-21). The PEGylation process attaches repeating units of ethylene
glycol (polyethylene glycol (PEG)) to a polypeptide drug. PEG molecules
have a large hydrodynamic volume (5-10 times the size of globular
proteins), are highly water soluble and hydrated, non-toxic,
non-immunogenic and rapidly cleared from the body. PEGylation of
molecules can lead to increased resistance of drugs to enzymatic
degradation, increased half-life in vivo, reduced dosing frequency,
decreased immunogenicity, increased physical and thermal stability,
increased solubility, increased liquid stability, and reduced
aggregation. The first PEGylated drugs were approved by the FDA in the
early 1990s. Since then, the FDA has approved several PEGylated drugs for
oral, injectable, and topical administration.

[0003] Polysialic acid (PSA), also referred to as colominic acid (CA), is
a naturally occurring polysaccharide. It is a homopolymer of
N-acetylneuraminic acid with α(2→8) ketosidic linkage and
contains vicinal diol groups at its non-reducing end. It is negatively
charged and a natural constituent of the human body. It can easily be
produced from bacteria in large quantities and with pre-determined
physical characteristics (U.S. Pat. No. 5,846,951). Because the
bacterially-produced PSA is chemically and immunologically identical to
PSA produced in the human body, bacterial PSA is non-immunogenic, even
when coupled to proteins. Unlike some polymers, PSA acid is
biodegradable. Covalent coupling of colominic acid to catalase and
asparaginase has been shown to increase enzyme stability in the presence
of proteolytic enzymes or blood plasma. Comparative studies in vivo with
polysialylated and unmodified asparaginase revealed that polysialylation
increased the half-life of the enzyme (Fernandes and Gregoriadis, Int J
Pharm. 2001; 217:215-24).

[0004] Coupling of PEG-derivatives to peptides or proteins is reviewed by
Roberts et al. (Adv Drug Deliv Rev 2002; 54:459-76). One approach for
coupling water soluble polymers to therapeutic proteins is the
conjugation of the polymers via the carbohydrate moieties of the protein.
Vicinal hydroxyl (OH) groups of carbohydrates in proteins can be easily
oxidized with sodium periodate (NaIO4) to form active aldehyde groups
(Rothfus et Smith, J Biol Chem 1963; 238:1402-10; van Lenten et Ashwell,
J Biol Chem 1971; 246:1889-94). Subsequently the polymer can be coupled
to the aldehyde groups of the carbohydrate by use of reagents containing,
for example, an active hydrazide group (Wilchek M and Bayer E A, Methods
Enzymol 1987; 138:429-42). A more recent technology is the use of
reagents containing aminooxy groups which react with aldehydes to form
oxime linkages (WO 96/40662, WO2008/025856).

[0005] Additional examples describing conjugation of a water soluble
polymer to a therapeutic protein are described in WO 06/071801 which
teaches the oxidation of carbohydrate moieties in Von Willebrand factor
and subsequent coupling to PEG using hydrazide chemistry; US Publication
No. 2009/0076237 which teaches the oxidation of rFVIII and subsequent
coupling to PEG and other water soluble polymers (e.g. PSA, HES, dextran)
using hydrazide chemistry; WO 2008/025856 which teaches oxidation of
different coagulation factors, e.g. rFIX, FVIII and FVIIa and subsequent
coupling to e.g., PEG, using aminooxy chemistry by forming an oxime
linkage; and U.S. Pat. No. 5,621,039 which teaches the oxidation of FIX
and subsequent coupling to PEG using hydrazide chemistry.

[0007] Although aniline catalysis can accelerate the oxime ligation
allowing short reaction times and the use of low concentrations of the
aminooxy reagent, aniline has toxic properties that must be considered
when, for example, the conjugated therapeutic protein to form the basis
of a pharmaceutical. For example, aniline has been shown to induce
methemoglobinemia (Harrison, J. H., and Jollow, D. J., Molecular
Pharmacology, 32(3) 423-431, 1987). Long-term dietary treatment of rats
has been shown to induce tumors in the spleen (Goodman, DG., et al., J
Natl Cancer Inst., 73(1):265-73, 1984). In vitro studies have also shown
that aniline has the potential to induce chromosome mutations and has the
potentially genotoxic activity (Bombhard E. M. et Herbold B, Critical
Reviews in Toxicology 35,783-835, 2005).

[0008] Considering the potentially dangerous properties of aniline and
notwithstanding the methods available of conjugating water soluble
polymers to therapeutic proteins, there remains a need to develop
materials and methods for conjugating water soluble polymers to proteins
that improves the protein's pharmacodynamic and/or pharmacokinetic
properties while minimizing the costs associated with the various
reagents and minimizing the health risks to the patient recipient.

SUMMARY OF THE INVENTION

[0009] The present invention provides materials and methods for
conjugating polymers to proteins that improves the protein's
pharmacodynamic and/or pharmacokinetic properties while minimizing the
costs associated with the various reagents and the health risks to the
patient recipients when the conjugation reaction is catalyzed by a
nucleophilic catalyst. In various embodiments of the invention,
alternative catalysts to substitute for aniline are provided.

[0012] In still another embodiment, an aforementioned method is provided
wherein a solution comprising an initial concentration of the therapeutic
protein between about 0.3 mg/ml and about 3.0 mg/ml is adjusted to a pH
value between about 5.0 and about 8.0 prior to contacting with the
activated water soluble polymer.

[0013] As used herein, the term "about" means a value above or below a
stated value. In various embodiments, the term "about" includes the
stated value plus or minus 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of the stated value.

[0014] In yet another embodiment, an aforementioned method is provided
wherein the initial concentration of the therapeutic protein is about 1.0
mg/ml and the pH is about 6.0. In a related embodiment, the initial
concentration of the therapeutic protein is about 0.75 mg/ml and the pH
is about 6.0. In still another related embodiment, the initial
concentration of the therapeutic protein is about 1.25 mg/ml and the pH
is about 6.0.

[0015] In another embodiment, an aforementioned method is provided wherein
the therapeutic protein is contacted by a desired excess concentration of
activated water soluble polymer, wherein the excess concentration is
between about 1-molar and about 300-molar excess. In another embodiment,
the excess concentration is about 50-fold molar excess.

[0016] In still another embodiment, an aforementioned method is provided
wherein the therapeutic protein is incubated with the activated water
soluble polymer under conditions comprising a time period between about
0.5 hours and about 24 hours; a temperature between about 2° C.
and about 37° C.; in the presence or absence of light; and with or
without stirring. In another embodiment, the conditions comprise a time
period of about 120 minutes, a temperature of about 22° C., the
absence of light; and with stirring. As used herein, the term "stirring"
is meant to include stirring at various speeds and intensities (e.g.,
gentle stirring) by commonly used laboratory or manufacturing equipment
and products.

[0017] In another embodiment, an aforementioned method is provided wherein
the nucleophilic catalyst is added in an amount to result in a final
concentration between about 1.0 mM and about 50 mM nucleophilic catalyst,
under conditions comprising a time period between about 0.1 minutes and
about 30 minutes; a temperature between about 2° C. and about
37° C.; in the presence or absence of light; and with or without
stirring. In another embodiment, the final concentration of the
nucleophilic catalyst is about 10 mM, and the conditions comprise a time
period of up to about 15 minutes, a temperature of about 22° C.,
the absence of light; and with stirring.

[0018] In still another embodiment, an aforementioned method is provided
wherein the oxidizing agent is added in an amount to result in a final
concentration between about 50 μM and about 1000 μM oxidizing
agent, under conditions comprising a time period between about 0.1
minutes and 120 minutes; a temperature between about 2° C. and
about 37° C.; in the presence or absence of light; and with or
without stirring. In another embodiment, the final concentration of
oxidizing agent is about 400 μM, and the conditions comprise a time
period of about 10 minutes, a temperature of about 22° C., the
absence of light and with stirring.

[0019] In yet another embodiment, an aforementioned method is provided
wherein the conjugating the water soluble polymer to the oxidized
carbohydrate moiety of the therapeutic protein is stopped by the addition
of a quenching agent selected from the group consisting of L-cysteine,
methionine, glutathione, glycerol, sodium meta bisulfite (Na2S2O5),
tryptophane, tyrosine, histidine or derivatives thereof, kresol,
imidazol, and combinations thereof; wherein the quenching agent is added
in an amount to result in a final concentration between about 1 mM and
about 100 mM quenching agent, under conditions comprising a time period
between about 5 minutes and about 120 minutes; a temperature between
about 2° C. and about 37° C.; in the presence or absence of
light; and with or without stirring. In another embodiment, the quenching
agent is L-cysteine. In still another embodiment, the L-cysteine is added
to result in a final concentration of about 10 mM and the conditions
comprise a time period of about 60 minutes, a temperature of about
22° C., the absence of light and with stirring.

[0020] In another embodiment, an aforementioned method is provided
comprising: a) a first step comprising adjusting the pH value of a
solution comprising the therapeutic protein to a pH value between about
5.0 and about 8.0, wherein the therapeutic protein concentration is
between about 0.3 mg/ml and about 3.0 mg/ml; b) a second step comprising
oxidizing one or more carbohydrates on the therapeutic protein, wherein
the oxidizing agent is added to the solution in the first step to result
in a final concentration between about 50 μM and about 1000 μM,
under conditions comprising a time period between about 0.1 minutes and
about 120 minutes; a temperature between about 2° C. and about
37° C.; in the presence or absence of light, and with or without
stirring; c) a third step comprising contacting the therapeutic protein
with a desired excess concentration of activated water soluble polymer,
wherein the excess concentration is between about 1-molar excess and
about 300-molar excess, under conditions comprising a time period between
about 0.5 hours and about 24 hours, a temperature between about 2°
C. and about 37° C.; in the presence or absence of light; and with
or without stirring; d) a fourth step comprising adding a nucleophilic
catalyst to the solution of the third step, wherein the nucleophilic
catalyst is added to result in a final concentration between about 1 mM
and about 50 mM, under conditions comprising a time period between about
0.1 minutes and about 30 minutes; a temperature between about 2°
C. and about 37° C.; in the presence or absence of light, and with
or without stirring; e) a fifth step wherein the therapeutic protein is
incubated with the activated water soluble polymer and nucleophilic
catalyst under conditions that allow conjugation of the activated
water-soluble polymer to one or more oxidized carbohydrates on the
therapeutic protein, said conditions comprising a time period between
about 0.5 hours and about 24 hours, a temperature between about 2°
C. and about 37° C.; in the presence or absence of light, and with
or without stirring; and f) a sixth step wherein the conjugating the
water soluble polymer to the one or more oxidized carbohydrates of the
therapeutic protein in the fifth step is stopped by the addition of a
quenching agent selected from the group consisting of L-cysteine,
methionine, glutathione, glycerol, Na2S2O5 (sodium meta bisulfite),
tryptophane, tyrosine, histidine or derivatives thereof, kresol,
imidazol, and combinations thereof; wherein the quenching agent is added
to result in a final concentration of about 1 mM and about 100 mM, under
conditions comprising a time period between about 5 minutes and about 120
minutes; a temperature between about 2° C. and about 37°
C.; in the presence or absence of light, and with or without stirring. In
another embodiment, the initial concentration of the therapeutic protein
in the first step is about 1 mg/ml and the pH is about 6.0; wherein the
final concentration of oxidizing agent in the second step is about 400
μM, and the conditions in the fifth step comprise a time period of
about 10 minutes, a temperature of about 22° C., the absence of
light and with stirring; wherein the excess concentration in the third
step is about 50 molar excess; wherein the conditions in the third step
comprise a time period of about 15 minutes, a temperature of about
22° C., the absence of light and with stirring; wherein the final
concentration of the nucleophilic catalyst in the fourth step is about 10
mM, and the conditions in the fourth step comprise a time period of about
15 minutes, a temperature of about 22° C., the absence of light
and with stirring; wherein the conditions of incubating the therapeutic
protein with the activated water soluble polymer and nucleophilic
catalyst in the fifth step comprise a time period of about 2 hours; a
temperature of about 22° C.; the absence of light; and with
stirring; and wherein the quenching agent in the sixth step is
L-cysteine; and wherein the L-cysteine is added to result in a final
concentration of about 10 mM and the conditions in the sixth step
comprise a time period of about 60 minutes, a temperature of about
22° C., the absence of light and with stirring.

[0021] In another embodiment, an aforementioned method is provided wherein
the water soluble polymer is PSA. In another embodiment the PSA is
comprised of about 10-300 sialic acid units. In another embodiment, the
water soluble polymer is PEG. In another embodiment, the water soluble
polymer is HES. In still another embodiment, the water soluble polymer is
HAS.

[0022] In still another embodiment, an aforementioned method is provided
wherein the therapeutic protein is FIX. In another embodiment, the
therapeutic protein is FVIIa. In another embodiment, the therapeutic
protein is FVIII.

[0023] In yet another embodiment, an aforementioned method is provided
wherein the oxidizing agent is sodium periodate (NaIO4).

[0024] In another embodiment, an aforementioned method is provided wherein
the oxidized carbohydrate moiety of the therapeutic protein is located in
the activation peptide of the blood coagulation protein.

[0025] In one embodiment, an aforementioned method is provided wherein PSA
is prepared by reacting an activated aminooxy linker with oxidized PSA;
wherein the aminooxy linker is selected from the group consisting of:
[0026] a) a 3-oxa-pentane-1,5-dioxyamine linker of the formula:

[0026] ##STR00001## [0027] b) a 3,6,9-trioxa-undecane-1,11-dioxyamine
linker of the formula:

##STR00002##

[0027] and [0028] c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine
linker of the formula:

##STR00003##

[0029] wherein the PSA is oxidized by incubation with a oxidizing agent to
form a terminal aldehyde group at the non-reducing end of the PSA. In a
related embodiment, the aminooxy linker is 3-oxa-pentane-1,5-dioxyamine.

[0030] In still another embodiment, an aforementioned method is provided
wherein the oxidizing agent is NaIO4.

[0031] In another embodiment, an aforementioned method is provided wherein
the nucleophilic catalyst is provided at a concentration between about 1
mM and about 50 mM. In one embodiment, the nucleophilic catalyst is
m-toluidine. In still another embodiment, the m-toluidine is present in
the conjugation reaction at a concentration of about 10 mM.

[0032] In yet another embodiment, an aforementioned method is provided
further comprising the step of reducing an oxime linkage in the
conjugated therapeutic protein by incubating the conjugated therapeutic
protein in a buffer comprising a reducing compound selected from the
group consisting of sodium cyanoborohydride (NaCNBH3), ascorbic acid
(vitamin C) and NaBH3. In one embodiment, the reducing compound is sodium
cyanoborohydride (NaCNBH3).

[0033] In still another embodiment, an aforementioned method is provided
further comprising the step of purifying the conjugated therapeutic
protein. In another embodiment, the conjugated therapeutic protein is
purified by a method selected from the group consisting of
chromatography, filtration and precipitation. In another embodiment, the
chromatography is selected from the group consisting of Hydrophobic
Interaction Chromatography (HIC), Ion Exchange chromatography (IEC), Size
exclusion chromatography (SEC), Affinity chromatography, and
Reversed-phase chromatography. In still another embodiment, an
anti-chaotropic salt is used in a chromatography loading step and in a
chromatography washing step. In yet another embodiment, the
chromatography takes place in a column. In another embodiment, the column
comprises a chromatography resin selected from the group consisting of
Phenyl-Sepharose FF and Butyl-Sepharose FF. In another embodiment, the
resin is present in the column at a bed height of between about 5 cm and
about 20 cm. In one embodiment, the bed height is about 10 cm.

[0034] In another embodiment, an aforementioned method is provided
comprising one or more washing steps wherein flow direction is set to
up-flow and wherein the flow rate is between about 0.2 cm/min and about
6.7 cm/min. As used herein, the term "down-flow" refers to a flow
direction from the top of the chromatographic column to the bottom of the
chromatographic column (normal flow direction/standard mode). As used
herein, the term "up-flow" refers to a flow direction from the bottom to
the top of the column (reversed flow direction). In one embodiment, the
flow rate is about 2 cm/min.

[0035] In another embodiment, an aforementioned method is provided
comprising one or more elution steps wherein flow direction is set to
down-flow and wherein the flow rate is between about 0.1 cm/min and about
6.7 cm/min. In a related embodiment, the flow rate is about 1 cm/min.

[0036] In still another embodiment, an aforementioned method is provided
comprising concentrating the conjugated therapeutic protein by
ultra-/diafiltration (UF/DF). In another embodiment, the final
concentration of therapeutic protein is between about 0.5 and about 3
mg/ml.

[0037] In another embodiment, an aforementioned method is provided wherein
the therapeutic protein comprises between about 5 and about 11
water-soluble polymer moieties. In another embodiment, the therapeutic
protein comprises between about 1 and about 3 water-soluble polymers.

[0038] In still another embodiment, an aforementioned method is provided
wherein the conjugated therapeutic protein is purified using
chromatography; wherein an anti-chaotropic salt is used for a loading
step and for a washing step; the method comprising one or more washing
steps wherein flow direction is set to up-flow and wherein the flow rate
is between about 0.2 cm/min and about 6.7 cm/min and one or more elution
steps wherein flow direction is set to down-flow and wherein the flow
rate is between about 0.2 cm/min and about 6.7 cm/min; further comprising
concentrating the conjugated therapeutic protein by ultra-/diafiltration
(UF/DF). In another embodiment, the chromatography is hydrophobic
interaction chromatography (HIC); wherein the one or more washing steps
flow rate is about 2 cm/min; and wherein the one or more elution steps
flow rate is about 1 cm/min.

[0039] In another embodiment, a modified therapeutic protein produced by
any of the aforementioned methods is provided.

[0044] In another embodiment, an aforementioned method is provided wherein
the water soluble polymer containing an active aminooxy group is prepared
by a method comprising: incubating a solution comprising an oxidized
water-soluble polymer with an activated aminooxy linker comprising an
active aminooxy group under conditions that allow the formation of a
stable oxime linkage between the oxidized water-soluble polymer and the
activated aminooxy linker, said conditions comprising a time period
between about 1 minute and about 24 hours; a temperature between about
2° C. and about 37° C.; in the presence or absence of
light, and with or without stirring; thereby forming a water soluble
polymer containing an active aminooxy group; and b) purifying the water
soluble polymer containing an active aminooxy group by a method selected
from the group consisting of chromatography, filtration and
precipitation. The term "activated water-soluble polymer" referes, in one
embodiment, to a water-soluble polyer containing an aldehyde group.

[0045] In yet another embodiment, an aforementioned method is provided
wherein the water soluble polymer containing an active aminooxy group is
prepared by a method comprising: a) incubating a solution comprising an
oxidized water-soluble polymer with an activated aminooxy linker
comprising an active aminooxy group under conditions that allow the
formation of a stable oxime linkage between the oxidized water-soluble
polymer and the activated aminooxy linker, said conditions comprising a
time period between about 1 minute and about 24 hours; a temperature
between about 2° C. and about 37° C.; in the presence or
absence of light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating a
solution comprising the water soluble polymer containing an active
aminooxy group of step a) with a reducing agent under conditions that
allow the formation of a stable alkoxamine linkage between the oxidized
water-soluble polymer and the activated aminooxy linker, said conditions
comprising a time period between about 1 minute and about 24 hours; a
temperature between about 2° C. and about 37° C.; in the
presence or absence of light; and with or without stirring; and c)
purifying the water soluble polymer containing an active aminooxy group
by a method selected from the group consisting of chromatography,
filtration and precipitation.

[0046] In still another embodiment, an aforementioned method is provided
wherein the water soluble polymer containing an active aminooxy group is
prepared by a method comprising: a) incubating a solution comprising an
oxidized water-soluble polymer with an activated aminooxy linker
comprising an active aminooxy group under conditions that allow the
formation of a stable oxime linkage between the oxidized water-soluble
polymer and the activated aminooxy linker, said conditions comprising a
time period between about 1 minute and about 24 hours; a temperature
between about 2° C. and about 37° C.; in the presence or
absence of light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating a
solution comprising the water soluble polymer containing an active
aminooxy group of step a) with a nucleophilic catalyst under conditions
comprising a time period between 1 minute and 24 hours; a temperature
between 2° C. and 37° C.; in the presence or absence of
light; and with or without stirring; and c) purifying the water soluble
polymer containing an active aminooxy group by a method selected from the
group consisting of chromatography, filtration and precipitation.

[0047] In yet another embodiment, an aforementioned method is provided
wherein the water soluble polymer containing an active aminooxy group is
prepared by a method comprising: a) incubating a solution comprising an
oxidized water-soluble polymer with an activated aminooxy linker
comprising an active aminooxy group under conditions that allow the
formation of a stable oxime linkage between the oxidized water-soluble
polymer and the activated aminooxy linker, said conditions comprising a
time period between about 1 minute and about 24 hours; a temperature
between about 2° C. and about 37° C.; in the presence or
absence of light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating a
solution comprising the water soluble polymer containing an active
aminooxy group of step a) with a nucleophilic catalyst under conditions
comprising a time period between 1 minute and 24 hours; a temperature
between 2° C. and 37° C.; in the presence or absence of
light; and with or without stirring; c) incubating a solution comprising
the water soluble polymer containing an active aminooxy group of step b)
with a reducing agent under conditions that allow the formation of a
stable alkoxamine linkage between the oxidized water-soluble polymer and
the activated aminooxy linker., said conditions comprising a time period
between about 1 minute and about 24 hours; a temperature between about
2° C. and about 37° C.; in the presence or absence of
light; and with or without stirring; and d) purifying the water soluble
polymer containing an active aminooxy group by a method selected from the
group consisting of chromatography, filtration and precipitation.

[0049] In another embodiment, an aforementioned method is provided wherein
the oxidizing agent is NaIO4.

[0050] In still another embodiment, an aforementioned method is provided
wherein the aminooxy linker is selected from the group consisting of:
[0051] a) a 3-oxa-pentane-1,5-dioxyamine linker of the formula:

[0051] ##STR00004## [0052] b) a 3,6,9-trioxa-undecane-1,11-dioxyamine
linker of the formula:

##STR00005##

[0052] and [0053] c) a 3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine
linker of the formula:

##STR00006##

[0054] In yet another embodiment, an aforementioned method is provided
wherein the reducing agent is selected from the group consisting of
sodium cyanoborohydride (NaCNBH3), ascorbic acid (vitamin C) and NaBH3.
In one embodiment, the reducing agent is sodium cyanoborohydride
(NaCNBH3).

[0055] In another embodiment, an aforementioned method is provided wherein
the nucleophilic catalyst is selected from the group consisting of
o-amino benzoic acid, m-amino benzoic acid, p-amino benzoic acid,
sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine, p-toluidine,
o-anisidine, m-anisidine, and p-anisidine. In one embodiment, the
nucleophilic catalyst is m-toluidine. In another embodiment, the
nucleophilic catalyst is added in an amount to result in a final
concentration between about 1.0 mM and about 50 mM nucleophilic catalyst.

[0056] In another embodiment, an aforementioned method is provided further
comprising concentrating the conjugated therapeutic protein by
ultra-/diafiltration (UF/DF).

[0057] In another embodiment, a method of conjugating a water soluble
polymer to an oxidized carbohydrate moiety of a blood coagulation protein
is provided comprising contacting the oxidized carbohydrate moiety with
an activated water soluble polymer under conditions that allow
conjugation;

[0060] said carbohydrate moiety oxidized by incubation with a buffer
comprising an oxidizing agent selected from the group consisting of
sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and potassium
perruthenate (KRuO4); wherein an oxime linkage is formed between the
oxidized carbohydrate moiety and the active aminooxy group on the water
soluble polymer.

[0063]FIG. 3 shows the synthesis of the water soluble di-aminoxy linkers
3-oxa-pentane-1,5-dioxyamine and 3,6,9-trioxa-undecane-1,11-dioxyamine.

[0064] FIG. 4 shows the preparation of aminooxy-PSA.

[0065] FIG. 5 shows the visualization of PSA-FIX conjugates prepared in
the presence of different catalysts by SDS PAGE. a) Comparison of aniline
with m-toluidine using different concentrations; b) Comparison of aniline
with o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid,
p-aminobenzamide and sulfanilic acid; c) Comparison of aniline and
m-toluidine with o-anisidine and m-anisidine.

[0067] The pharmacological and immunological properties of therapeutic
proteins can be improved by chemical modification and conjugation with
polymeric compounds such as polyethylene glycol (PEG), branched PEG,
polysialic acid (PSA), hydroxyalkyl starch (HAS), hydroxylethyl starch
(HES), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic
acid, chondroitin sulfate, dermatan sulfate, starch, dextran,
carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol
(PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine,
polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone,
polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(1-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC). The
properties of the resulting conjugates generally strongly depend on the
structure and the size of the polymer. Thus, polymers with a defined and
narrow size distribution are usually preferred in the art. Synthetic
polymers like PEG can be manufactured easily with a narrow size
distribution, while PSA can be purified in such a manner that results in
a final PSA preparation with a narrow size distribution. In addition
PEGylation reagents with defined polymer chains and narrow size
distribution are on the market and commercially available for a
reasonable price.

[0068] The addition of a soluble polymer, such as through polysialylation,
is one approach to improve the properties of therapeutic proteins such as
the blood coagulation protein FIX, as well as other coagulation proteins
(e.g., VWF, FVIIa (see, e.g., US 2008/0221032A1, incorporated herein by
reference) and FVIII).

Therapeutic Proteins

[0069] In certain embodiments of the invention, the aforementioned
polypeptides and polynucleotides are exemplified by the following
therapeutic proteins: enzymes, antigens, antibodies, receptors, blood
coagulation proteins, growth factors, hormones, and ligands. In certain
embodiments, the therapeutic protein is a blood coagulation protein such
as Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), Von
Willebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI (FXI),
Factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1,
tissue factor (TF) or ADAMTS 13 protease. In one embodiment, a
therapeutic protein according to the invention is a glycoprotein or, in
various embodiments, a protein that is not naturally glycosylated in vivo
(i.e., a protein that does not contain a natural glycosylation site or a
protein that is not glycosylated in a host cell prior to purification).

[0072] The therapeutic proteins provided herein should not be considered
to be exclusive. Rather, as is apparent from the disclosure provided
herein, the methods of the invention are applicable to any protein
wherein attachment of a water soluble polymer is desired according to the
invention. For example, therapeutic proteins are described in US
2007/0026485, incorporated herein by reference in its entirety.

Blood Coagulation Proteins

[0073] In one aspect, the starting material of the present invention is a
blood coagulation protein, which can be derived from human plasma, or
produced by recombinant engineering techniques, as described in U.S. Pat.
No. 4,757,006; U.S. Pat. No. 5,733,873; U.S. Pat. No. 5,198,349; U.S.
Pat. No. 5,250,421; U.S. Pat. No. 5,919,766; and EP 306 968.

[0076] The blood coagulation cascade is divided into three distinct
segments: the intrinsic, extrinsic, and common pathways (Schenone et al.,
Curr Opin Hematol. 2004; 11:272-7). The cascade involves a series of
serine protease enzymes (zymogens) and protein cofactors. When required,
an inactive zymogen precursor is converted into the active form, which
consequently converts the next enzyme in the cascade.

[0078] The extrinsic pathway is initiated when the vascular lumen of blood
vessels is damaged. The membrane glycoprotein tissue factor is exposed
and then binds to circulating factor VII (FVII) and to small preexisting
amounts of its activated form FVIIa. This binding facilitates full
conversion of FVII to FVIIa and subsequently, in the presence of calcium
and phospholipids, the conversion of factor IX (FIX) to factor IXa (FIXa)
and factor X (FX) to factor Xa (FXa). The association of FVIIa with
tissue factor enhances the proteolytic activity by bringing the binding
sites of FVII for the substrate (FIX and FX) into closer proximity and by
inducing a conformational change, which enhances the enzymatic activity
of FVIIa.

[0079] The activation of FX is the common point of the two pathways. Along
with phospholipid and calcium, factors Va (FVa) and Xa convert
prothrombin to thrombin (prothrombinase complex), which then cleaves
fibrinogen to form fibrin monomers. The monomers polymerize to form
fibrin strands. Factor XIIIa (FXIIIa) covalently bonds these strands to
one another to form a rigid mesh.

[0080] Conversion of FVII to FVIIa is also catalyzed by a number of
proteases, including thrombin, FIXa, FXa, factor XIa (FXIa), and factor
XIIa (FXIIa). For inhibition of the early phase of the cascade, tissue
factor pathway inhibitor targets FVIIa/tissue factor/FXa product complex.

Factor VIIa

[0081] FVII (also known as stable factor or proconvertin) is a vitamin
K-dependent serine protease glycoprotein with a pivotal role in
hemostasis and coagulation (Eigenbrot, Curr Protein Pept Sci. 2002;
3:287-99).

[0082] FVII is synthesized in the liver and secreted as a single-chain
glycoprotein of 48 kD. FVII shares with all vitamin K-dependent serine
protease glycoproteins a similar protein domain structure consisting of
an amino-terminal gamma-carboxyglutamic acid (Gla) domain with 9-12
residues responsible for the interaction of the protein with lipid
membranes, a carboxy-terminal serine protease domain (catalytic domain),
and two epidermal growth factor-like domains containing a calcium ion
binding site that mediates interaction with tissue factor. Gamma-glutamyl
carboxylase catalyzes carboxylation of Gla residues in the amino-terminal
portion of the molecule. The carboxylase is dependent on a reduced form
of vitamin K for its action, which is oxidized to the epoxide form.
Vitamin K epoxide reductase is required to convert the epoxide form of
vitamin K back to the reduced form.

[0083] The major proportion of FVII circulates in plasma in zymogen form,
and activation of this form results in cleavage of the peptide bond
between arginine 152 and isoleucine 153. The resulting activated FVIIa
consists of a NH2-derived light chain (20 kD) and a COOH terminal-derived
heavy chain (30 kD) linked via a single disulfide bond (Cys 135 to Cys
262). The light chain contains the membrane-binding Gla domain, while the
heavy chain contains the catalytic domain.

[0084] The plasma concentration of FVII determined by genetic and
environmental factors is about 0.5 mg/mL (Pinotti et al., Blood. 2000;
95:3423-8). Different FVII genotypes can result in several-fold
differences in mean FVII levels. Plasma FVII levels are elevated during
pregnancy in healthy females and also increase with age and are higher in
females and in persons with hypertriglyceridemia. FVII has the shortest
half-life of all procoagulant factors (3-6 h). The mean plasma
concentration of FVIIa is 3.6 ng/mL in healthy individuals and the
circulating half-life of FVIIa is relatively long (2.5 h) compared with
other coagulation factors.

[0085] Hereditary FVII deficiency is a rare autosomal recessive bleeding
disorder with a prevalence estimated to be 1 case per 500,000 persons in
the general population (Acharya et al., J Thromb Haemost. 2004; 2248-56).
Acquired FVII deficiency from inhibitors is also very rare. Cases have
also been reported with the deficiency occurring in association with
drugs such as cephalosporins, penicillins, and oral anticoagulants.
Furthermore, acquired FVII deficiency has been reported to occur
spontaneously or with other conditions, such as myeloma, sepsis, aplastic
anemia, with interleukin-2 and antithymocyte globulin therapy.

[0087] FIX is a vitamin K-dependent plasma protein that participates in
the intrinsic pathway of blood coagulation by converting FX to its active
form in the presence of calcium ions, phospholipids and FVIIIa. The
predominant catalytic capability of FIX is as a serine protease with
specificity for a particular arginine-isoleucine bond within FX.
Activation of FIX occurs by FXIa which causes excision of the activation
peptide from FIX to produce an activated FIX molecule comprising two
chains held by one or more disulphide bonds. Defects in FIX are the cause
of recessive X-linked hemophilia B.

[0088] Hemophilia A and B are inherited diseases characterized by
deficiencies in FVIII and FIX polypeptides, respectively. The underlying
cause of the deficiencies is frequently the result of mutations in FVIII
and FIX genes, both of which are located on the X chromosome. Traditional
therapy for hemophilias often involves intravenous administration of
pooled plasma or semi-purified coagulation proteins from normal
individuals. These preparations can be contaminated by pathogenic agents
or viruses, such as infectious prions, HIV, parvovirus, hepatitis A, and
hepatitis C. Hence, there is an urgent need for therapeutic agents that
do not require the use of human serum.

[0089] The level of the decrease in FIX activity is directly proportional
to the severity of hemophilia B. The current treatment of hemophilia B
consists of the replacement of the missing protein by plasma-derived or
recombinant FIX (so-called FIX substitution or replacement treatment or
therapy).

[0090] Polynucleotide and polypeptide sequences of FIX can be found for
example in the UniProtKB/Swiss-Prot Accession No. P00740, U.S. Pat. No.
6,531,298 and in FIG. 1 (SEQ ID NO: 1).

Factor VIII

[0091] Coagulation factor VIII (FVIII) circulates in plasma at a very low
concentration and is bound non-covalently to Von Willebrand factor (VWF).
During hemostasis, FVIII is separated from VWF and acts as a cofactor for
activated factor IX (FIXa)-mediated FX activation by enhancing the rate
of activation in the presence of calcium and phospholipids or cellular
membranes.

[0092] FVIII is synthesized as a single-chain precursor of approximately
270-330 kD with the domain structure A I-A2-B-A3-C1-C2. When purified
from plasma (e.g., "plasma-derived" or "plasmatic"), FVIII is composed of
a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The molecular mass
of the light chain is 80 kD whereas, due to proteolysis within the B
domain, the heavy chain is in the range of 90-220 kD.

[0093] FVIII is also synthesized as a recombinant protein for therapeutic
use in bleeding disorders. Various in vitro assays have been devised to
determine the potential efficacy of recombinant FVIII (rFVIII) as a
therapeutic medicine. These assays mimic the in vivo effects of
endogenous FVIII. In vitro thrombin treatment of FVIII results in a rapid
increase and subsequent decrease in its procoagulant activity, as
measured by in vitro assays. This activation and inactivation coincides
with specific limited proteolysis both in the heavy and the light chains,
which alter the availability of different binding epitopes in FVIII, e.g.
allowing FVIII to dissociate from VWF and bind to a phospholipid surface
or altering the binding ability to certain monoclonal antibodies.

[0094] The lack or dysfunction of FVIII is associated with the most
frequent bleeding disorder, hemophilia A. The treatment of choice for the
management of hemophilia A is replacement therapy with plasma derived or
rFVIII concentrates. Patients with severe hemophilia A with FVIII levels
below 1%, are generally on prophylactic therapy with the aim of keeping
FVIII above 1% between doses. Taking into account the average half-lives
of the various FVIII products in the circulation, this result can usually
be achieved by giving FVIII two to three times a week.

[0096] Von Willebrand factor (VWF) is a glycoprotein circulating in plasma
as a series of multimers ranging in size from about 500 to 20,000 kD.
Multimeric forms of VWF are composed of 250 kD polypeptide subunits
linked together by disulfide bonds. VWF mediates initial platelet
adhesion to the sub-endothelium of the damaged vessel wall. Only the
larger multimers exhibit hemostatic activity. It is assumed that
endothelial cells secrete large polymeric forms of VWF and those forms of
VWF which have a low molecular weight (low molecular weight VWF) arise
from proteolytic cleavage. The multimers having large molecular masses
are stored in the Weibel-Pallade bodies of endothelial cells and
liberated upon stimulation.

[0097] VWF is synthesized by endothelial cells and megakaryocytes as
prepro-VWF that consists to a large extent of repeated domains. Upon
cleavage of the signal peptide, pro-VWF dimerizes through disulfide
linkages at its C-terminal region. The dimers serve as protomers for
multimerization, which is governed by disulfide linkages between the free
end termini. The assembly to multimers is followed by the proteolytic
removal of the propeptide sequence (Leyte et al., Biochem. J. 274 (1991),
257-261).

[0099] Defects in VWF are causal to Von Willebrand disease (VWD), which is
characterized by a more or less pronounced bleeding phenotype. VWD type 3
is the most severe form, in which VWF is completely missing, and VWD type
1 relates to a quantitative loss of VWF and its phenotype can be very
mild. VWD type 2 relates to qualitative defects of VWF and can be as
severe as VWD type 3. VWD type 2 has many sub forms, some being
associated with the loss or the decrease of high molecular weight
multimers. Von Willebrand disease type 2a (VWD-2A) is characterized by a
loss of both intermediate and large multimers. VWD-2B is characterized by
a loss of highest-molecular-weight multimers. Other diseases and
disorders related to VWF are known in the art.

[0100] The polynucleotide and amino acid sequences of prepro-VWF are
available at GenBank Accession Nos. NM--000552 and NP--000543,
respectively.

[0101] Other blood coagulation proteins according to the present invention
are described in the art, e.g. Mann K G, Thromb Haemost, 1999; 82:165-74.

A. Polypeptides

[0102] In one aspect, the starting material of the present invention is a
protein or polypeptide. As described herein, the term therapeutic protein
refers to any therapeutic protein molecule which exhibits biological
activity that is associated with the therapeutic protein. In one
embodiment of the invention, the therapeutic protein molecule is a
full-length protein.

[0103] Therapeutic protein molecules contemplated include full-length
proteins, precursors of full length proteins, biologically active
subunits or fragments of full length proteins, as well as biologically
active derivatives and variants of any of these forms of therapeutic
proteins. Thus, therapeutic protein include those that (1) have an amino
acid sequence that has greater than about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%
or greater amino acid sequence identity, over a region of at least about
25, about 50, about 100, about 200, about 300, about 400, or more amino
acids, to a polypeptide encoded by a referenced nucleic acid or an amino
acid sequence described herein; and/or (2) specifically bind to
antibodies, e.g., polyclonal or monoclonal antibodies, generated against
an immunogen comprising a referenced amino acid sequence as described
herein, an immunogenic fragment thereof, and/or a conservatively modified
variant thereof.

[0104] According to the present invention, the term "recombinant
therapeutic protein" includes any therapeutic protein obtained via
recombinant DNA technology. In certain embodiments, the term encompasses
proteins as described herein.

[0105] As used herein, "endogenous therapeutic protein" includes a
therapeutic protein which originates from the mammal intended to receive
treatment. The term also includes therapeutic protein transcribed from a
transgene or any other foreign DNA present in said mammal. As used
herein, "exogenous therapeutic protein" includes a blood coagulation
protein which does not originate from the mammal intended to receive
treatment.

[0106] As used herein, "plasma-derived blood coagulation protein" or
"plasmatic" includes all forms of the protein found in blood obtained
from a mammal having the property participating in the coagulation
pathway.

[0107] As used herein "biologically active derivative" or "biologically
active variant" includes any derivative or variant of a molecule having
substantially the same functional and/or biological properties of said
molecule, such as binding properties, and/or the same structural basis,
such as a peptidic backbone or a basic polymeric unit.

[0108] An "analog," such as a "variant" or a "derivative," is a compound
substantially similar in structure and having the same biological
activity, albeit in certain instances to a differing degree, to a
naturally-occurring molecule. For example, a polypeptide variant refers
to a polypeptide sharing substantially similar structure and having the
same biological activity as a reference polypeptide. Variants or analogs
differ in the composition of their amino acid sequences compared to the
naturally-occurring polypeptide from which the analog is derived, based
on one or more mutations involving (i) deletion of one or more amino acid
residues at one or more termini of the polypeptide and/or one or more
internal regions of the naturally-occurring polypeptide sequence (e.g.,
fragments), (ii) insertion or addition of one or more amino acids at one
or more termini (typically an "addition" or "fusion") of the polypeptide
and/or one or more internal regions (typically an "insertion") of the
naturally-occurring polypeptide sequence or (iii) substitution of one or
more amino acids for other amino acids in the naturally-occurring
polypeptide sequence. By way of example, a "derivative" is a type of
analog and refers to a polypeptide sharing the same or substantially
similar structure as a reference polypeptide that has been modified,
e.g., chemically.

[0109] A variant polypeptide is a type of analog polypeptide and includes
insertion variants, wherein one or more amino acid residues are added to
a therapeutic protein amino acid sequence of the invention. Insertions
may be located at either or both termini of the protein, and/or may be
positioned within internal regions of the therapeutic protein amino acid
sequence. Insertion variants, with additional residues at either or both
termini, include for example, fusion proteins and proteins including
amino acid tags or other amino acid labels. In one aspect, the blood
coagulation protein molecule optionally contains an N-terminal Met,
especially when the molecule is expressed recombinantly in a bacterial
cell such as E. coli.

[0110] In deletion variants, one or more amino acid residues in a
therapeutic protein polypeptide as described herein are removed.
Deletions can be effected at one or both termini of the therapeutic
protein polypeptide, and/or with removal of one or more residues within
the therapeutic protein amino acid sequence. Deletion variants,
therefore, include fragments of a therapeutic protein polypeptide
sequence.

[0111] In substitution variants, one or more amino acid residues of a
therapeutic protein polypeptide are removed and replaced with alternative
residues. In one aspect, the substitutions are conservative in nature and
conservative substitutions of this type are well known in the art.
Alternatively, the invention embraces substitutions that are also
non-conservative. Exemplary conservative substitutions are described in
Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York
(1975), pp. 71-77] and are set out immediately below.

[0114] Polynucleotides encoding a therapeutic protein of the invention
also include, without limitation, those that (1) specifically hybridize
under stringent hybridization conditions to a nucleic acid encoding a
referenced amino acid sequence as described herein, and conservatively
modified variants thereof; (2) have a nucleic acid sequence that has
greater than about 95%, about 96%, about 97%, about 98%, about 99%, or
higher nucleotide sequence identity, over a region of at least about 25,
about 50, about 100, about 150, about 200, about 250, about 500, about
1000, or more nucleotides (up to the full length sequence of 1218
nucleotides of the mature protein), to a reference nucleic acid sequence
as described herein. Exemplary "stringent hybridization" conditions
include hybridization at 42° C. in 50% formamide, 5×SSC, 20
mM Na.PO4, pH 6.8; and washing in 1×SSC at 55° C. for 30
minutes. It is understood that variation in these exemplary conditions
can be made based on the length and GC nucleotide content of the
sequences to be hybridized. Formulas standard in the art are appropriate
for determining appropriate hybridization conditions. See Sambrook et
al., Molecular Cloning: A Laboratory Manual (Second ed., Cold Spring
Harbor Laboratory Press, 1989) §§9.47-9.51.

[0115] A "naturally-occurring" polynucleotide or polypeptide sequence is
typically derived from a mammal including, but not limited to, primate,
e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep,
or any mammal. The nucleic acids and proteins of the invention can be
recombinant molecules (e.g., heterologous and encoding the wild type
sequence or a variant thereof, or non-naturally occurring).

C. Production of Therapeutic Proteins

[0116] Production of a therapeutic protein includes any method known in
the art for (i) the production of recombinant DNA by genetic engineering,
(ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by,
for example and without limitation, transfection, electroporation or
microinjection, (iii) cultivating said transformed cells, (iv) expressing
therapeutic protein, e.g. constitutively or upon induction, and (v)
isolating said blood coagulation protein, e.g. from the culture medium or
by harvesting the transformed cells, in order to obtain purified
therapeutic protein.

[0117] In other aspects, the therapeutic protein is produced by expression
in a suitable prokaryotic or eukaryotic host system characterized by
producing a pharmacologically acceptable blood coagulation protein
molecule. Examples of eukaryotic cells are mammalian cells, such as CHO,
COS, HEK 293, BHK, SK-Hep, and HepG2.

[0118] A wide variety of vectors are used for the preparation of the
therapeutic protein and are selected from eukaryotic and prokaryotic
expression vectors. Examples of vectors for prokaryotic expression
include plasmids such as, and without limitation, pRSET, pET, and pBAD,
wherein the promoters used in prokaryotic expression vectors include one
or more of, and without limitation, lac, trc, trp, recA, or araBAD.
Examples of vectors for eukaryotic expression include: (i) for expression
in yeast, vectors such as, and without limitation, pAO, pPIC, pYES, or
pMET, using promoters such as, and without limitation, AOX1, GAP, GAL1,
or AUG1; (ii) for expression in insect cells, vectors such as and without
limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as and
without limitation PH, p10, MT, Ac5, OpIE2, gp64, or polh, and (iii) for
expression in mammalian cells, vectors such as and without limitation
pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one
aspect, viral systems such as and without limitation vaccinia virus,
adeno-associated viruses, herpes viruses, or retroviruses, using
promoters such as and without limitation CMV, SV40, EF-1, UbC, RSV, ADV,
BPV, and β-actin.

D. Administration

[0119] In one embodiment a conjugated therapeutic protein of the present
invention may be administered by injection, such as intravenous,
intramuscular, or intraperitoneal injection.

[0120] To administer compositions comprising a conjugated therapeutic
protein of the present invention to human or test animals, in one aspect,
the compositions comprise one or more pharmaceutically acceptable
carriers. The terms "pharmaceutically" or "pharmacologically acceptable"
refer to molecular entities and compositions that are stable, inhibit
protein degradation such as aggregation and cleavage products, and in
addition do not produce allergic, or other adverse reactions when
administered using routes well-known in the art, as described below.
"Pharmaceutically acceptable carriers" include any and all clinically
useful solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like, including
those agents disclosed above.

[0121] As used herein, "effective amount" includes a dose suitable for
treating a disease or disorder or ameliorating a symptom of a disease or
disorder. In one embodiment, "effective amount" includes a dose suitable
for treating a mammal having a bleeding disorder as described herein.

[0122] The compositions may be administered orally, topically,
transdermally, parenterally, by inhalation spray, vaginally, rectally, or
by intracranial injection. The term parenteral as used herein includes
subcutaneous injections, intravenous, intramuscular, intracisternal
injection, or infusion techniques. Administration by intravenous,
intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,
retrobulbar, intrapulmonary injection and or surgical implantation at a
particular site is contemplated as well. Generally, compositions are
essentially free of pyrogens, as well as other impurities that could be
harmful to the recipient.

[0123] Single or multiple administrations of the compositions can be
carried out with the dose levels and pattern being selected by the
treating physician. For the prevention or treatment of disease, the
appropriate dosage will depend on the type of disease to be treated, as
described above, the severity and course of the disease, whether drug is
administered for preventive or therapeutic purposes, previous therapy,
the patient's clinical history and response to the drug, and the
discretion of the attending physician.

[0124] The present invention also relates to a pharmaceutical composition
comprising an effective amount of a conjugated therapeutic protein as
defined herein. The pharmaceutical composition may further comprise a
pharmaceutically acceptable carrier, diluent, salt, buffer, or excipient.
The pharmaceutical composition can be used for treating the above-defined
bleeding disorders. The pharmaceutical composition of the invention may
be a solution or a lyophilized product. Solutions of the pharmaceutical
composition may be subjected to any suitable lyophilization process.

[0125] As an additional aspect, the invention includes kits which comprise
a composition of the invention packaged in a manner which facilitates its
use for administration to subjects. In one embodiment, such a kit
includes a compound or composition described herein (e.g., a composition
comprising a conjugated therapeutic protein), packaged in a container
such as a sealed bottle or vessel, with a label affixed to the container
or included in the package that describes use of the compound or
composition in practicing the method. In one embodiment, the kit contains
a first container having a composition comprising a conjugated
therapeutic protein and a second container having a physiologically
acceptable reconstitution solution for the composition in the first
container. In one aspect, the compound or composition is packaged in a
unit dosage form. The kit may further include a device suitable for
administering the composition according to a specific route of
administration. Preferably, the kit contains a label that describes use
of the therapeutic protein or peptide composition.

[0128] PSAs consist of polymers (generally homopolymers) of
N-acetylneuraminic acid. The secondary amino group normally bears an
acetyl group, but it may instead bear a glycolyl group. Possible
substituents on the hydroxyl groups include acetyl, lactyl, ethyl,
sulfate, and phosphate groups.

##STR00007##

[0129] Structure of Sialic Acid (N-acetylneuraminic acid)

[0130] PSAs and mPSAs generally comprise linear polymers consisting
essentially of N-acetylneuraminic acid moieties linked by 2,8- or
2,9-glycosidic linkages or combinations of these (e.g. alternating 2,8-
and 2,9-linkages). In particularly preferred PSAs and mPSAs, the
glycosidic linkages are α-2,8. Such PSAs and mPSAs are conveniently
derived from colominic acids, and are referred to herein as "CAs" and
"mCAs". Typical PSAs and mPSAs comprise at least 2, preferably at least
5, more preferably at least 10 and most preferably at least 20
N-acetylneuraminic acid moieties. Thus, they may comprise from 2 to 300
N-acetylneuraminic acid moieties, preferably from 5 to 200
N-acetylneuraminic acid moieties, or most preferably from 10 to 100
N-acetylneuraminic acid moieties. PSAs and CAs preferably are essentially
free of sugar moieties other than N-acetylneuraminic acid. Thus PSAs and
CAs preferably comprise at least 90%, more preferably at least 95% and
most preferably at least 98% N-acetylneuraminic acid moieties.

[0131] Where PSAs and CAs comprise moieties other than N-acetylneuraminic
acid (as, for example in mPSAS and mCAs) these are preferably located at
one or both of the ends of the polymer chain. Such "other" moieties may,
for example, be moieties derived from terminal N-acetylneuraminic acid
moieties by oxidation or reduction.

[0132] For example, WO-A-0187922 describes such mPSAs and mCAs in which
the non-reducing terminal N-acetylneuraminic acid unit is converted to an
aldehyde group by reaction with sodium periodate. Additionally, WO
2005/016974 describes such mPSAs and mCAs in which the reducing terminal
N-acetylneuraminic acid unit is subjected to reduction to reductively
open the ring at the reducing terminal N-acetylneuraminic acid unit,
whereby a vicinal diol group is formed, followed by oxidation to convert
the vicinal diol group to an aldehyde group.

[0133] Sialic acid rich glycoproteins bind selectin in humans and other
organisms. They play an important role in human influenza infections.
E.g., sialic acid can hide mannose antigens on the surface of host cells
or bacteria from mannose-binding lectin. This prevents activation of
complement. Sialic acids also hide the penultimate galactose residue thus
preventing rapid clearance of the glycoprotein by the galactose receptor
on the hepatic parenchymal cells.

[0135] Colominic acids (a sub-class of PSAs) are homopolymers of
N-acetylneuraminic acid (NANA) with a (2→8) ketosidic linkage, and
are produced, inter alia, by particular strains of Escherichia coli
possessing K1 antigen. Colominic acids have many physiological functions.
They are important as a raw material for drugs and cosmetics.

[0137] As used herein, "sialic acid moieties" includes sialic acid
monomers or polymers ("polysaccharides") which are soluble in an aqueous
solution or suspension and have little or no negative impact, such as
side effects, to mammals upon administration of the PSA-blood coagulation
protein conjugate in a pharmaceutically effective amount. The polymers
are characterized, in one aspect, as having 1, 2, 3, 4, 5, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 sialic acid units. In
certain aspects, different sialic acid units are combined in a chain.

[0138] In one embodiment of the invention, the sialic acid portion of the
polysaccharide compound is highly hydrophilic, and in another embodiment
the entire compound is highly hydrophilic. Hydrophilicity is conferred
primarily by the pendant carboxyl groups of the sialic acid units, as
well as the hydroxyl groups. The saccharide unit may contain other
functional groups, such as, amine, hydroxyl or sulphate groups, or
combinations thereof. These groups may be present on naturally-occurring
saccharide compounds, or introduced into derivative polysaccharide
compounds.

[0139] The naturally occurring polymer PSA is available as a polydisperse
preparation showing a broad size distribution (e.g. Sigma C-5762) and
high polydispersity (PD). Because the polysaccharides are usually
produced in bacteria carrying the inherent risk of copurifying
endotoxins, the purification of long sialic acid polymer chains may raise
the probability of increased endotoxin content. Short PSA molecules with
1-4 sialic acid units can also be synthetically prepared (Kang S H et
al., Chem Commun. 2000; 227-8; Ress D K and Linhardt R J, Current Organic
Synthesis. 2004; 1:31-46), thus minimizing the risk of high endotoxin
levels. However PSA preparations with a narrow size distribution and low
polydispersity, which are also endotoxin-free, can now be manufactured.
Polysaccharide compounds of particular use for the invention are, in one
aspect, those produced by bacteria. Some of these naturally-occurring
polysaccharides are known as glycolipids. In one embodiment, the
polysaccharide compounds are substantially free of terminal galactose
units.

B. Polyethylene Glycol (PEG) and Pegylation

[0140] In certain aspects, therapeutic proteins are conjugated to a water
soluble polymer by any of a variety of chemical methods (Roberts J M et
al., Advan Drug Delivery Rev 2002; 54:459-76). For example, in one
embodiment a therapeutic protein is modified by the conjugation of PEG to
free amino groups of the protein using N-hydroxysuccinimide (NHS) esters.
In another embodiment the water soluble polymer, for example PEG, is
coupled to free SH groups using maleimide chemistry or the coupling of
PEG hydrazides or PEG amines to carbohydrate moieties of the therapeutic
protein after prior oxidation.

[0142] In one embodiment of the invention, a therapeutic protein is
modified via lysine residues by use of polyethylene glycol derivatives
containing an active N-hydroxysuccinimide ester (NHS) such as
succinimidyl succinate, succinimidyl glutarate or succinimidyl
propionate. These derivatives react with the lysine residues of the
therapeutic protein under mild conditions by forming a stable amide bond.
In one embodiment of the invention, the chain length of the PEG
derivative is 5,000 Da. Other PEG derivatives with chain lengths of 500
to 2,000 Da, 2,000 to 5,000 Da, greater than 5,000 up to 10,000 Da or
greater than 10,000 up to 20,000 Da, or greater than 20,000 up to 150,000
Da are used in various embodiments, including linear and branched
structures.

[0143] Alternative methods for the PEGylation of amino groups are, without
limitation, the chemical conjugation with PEG carbonates by forming
urethane bonds, or the reaction with aldehydes or ketones by reductive
amination forming secondary amide bonds.

[0144] In one embodiment of the present invention a therapeutic protein
molecule is chemically modified using PEG derivatives that are
commercially available. These PEG derivatives in alternative aspects have
linear or branched structures. Examples of PEG-derivatives containing NHS
groups are listed below.

[0159] These propane derivatives show a glycerol backbone with a 1,2
substitution pattern. In the present invention branched PEG derivatives
based on glycerol structures with 1,3 substitution or other branched
structures described in US2003/0143596A1 are also contemplated.

[0161] Surprisingly, the PEGylated therapeutic protein of this invention
exhibits functional activity, combined with an extended half-life in
vivo. In addition the PEGylated rFVIII, FVIIa, FIX, or other blood
coagulation factor seems to be more resistant against thrombin
inactivation.

C. Hydroxyalkyl Starch (HAS) and Hydroxylethyl Starch (HES)

[0162] In various embodiments of the present invention, a therapeutic
protein molecule is chemically modified using hydroxyalkyl starch (HAS)
or hydroxylethyl starch (HES) or derivatives thereof.

[0163] HES is a derivative of naturally occurring amylopectin and is
degraded by alpha-amylase in the body. HES is a substituted derivative of
the carbo-hydrate polymer amylopectin, which is present in corn starch at
a concentration of up to 95% by weight. HES exhibits advantageous
biological properties and is used as a blood volume replacement agent and
in hemodilution therapy in the clinics (Sommermeyer et al., 1987,
Krankenhauspharmazie, 8 (8), 271-278; and Weidler et al., 1991,
Arzneim.-Forschung/Drug Res. g 419 494-498).

[0164] Amylopectin consists of glucose moieties, wherein in the main chain
alpha-1,4-glycosidic bonds are present and at the branching sites
alpha-1,6-glycosidic bonds are found. The physical-chemical properties of
this molecule are mainly determined by the type of glycosidic bonds. Due
to the nicked alpha-1,4-glycosidic bond, helical structures with about
six glucose-monomers per turn are produced. The physico-chemical as well
as the biochemical properties of the polymer can be modified via
substitution. The introduction of a hydroxyethyl group can be achieved
via alkaline hydroxyethylation. By adapting the reaction conditions it is
possible to exploit the different reactivity of the respective hydroxy
group in the unsubstituted glucose monomer with respect to a
hydroxyethylation. Owing to this fact, the skilled person is able to
influence the substitution pattern to a limited extent.

[0165] HAS refers to a starch derivative which has been substituted by at
least one hydroxyalkyl group. Therefore, the term hydroxyalkyl starch is
not limited to compounds where the terminal carbohydrate moiety comprises
hydroxyalkyl groups R1, R2, and/or R3, but also refers to compounds in
which at least one hydroxy group present anywhere, either in the terminal
carbohydrate moiety and/or in the remaining part of the starch molecule,
HAS', is substituted by a hydroxyalkyl group R1, R2, or R3.

##STR00020##

[0166] The alkyl group may be a linear or branched alkyl group which may
be suitably substituted. Preferably, the hydroxyalkyl group contains 1 to
10 carbon atoms, more preferably from 1 to 6 carbon atoms, more
preferably from 1 to 4 carbon atoms, and even more preferably 2-4 carbon
atoms. "Hydroxyalkyl starch" therefore preferably comprises hydroxyethyl
starch, hydroxypropyl starch and hydroxybutyl starch, wherein
hydroxyethyl starch and hydroxypropyl starch are particularly preferred.

[0167] Hydroxyalkyl starch comprising two or more different hydroxyalkyl
groups is also comprised in the present invention. The at least one
hydroxyalkyl group comprised in HAS may contain two or more hydroxy
groups. According to one embodiment, the at least one hydroxyalkyl group
comprised HAS contains one hydroxy group.

[0168] The term HAS also includes derivatives wherein the alkyl group is
mono- or polysubstituted. In one embodiment, the alkyl group is
substituted with a halogen, especially fluorine, or with an aryl group,
provided that the HAS remains soluble in water. Furthermore, the terminal
hydroxy group a of hydroxyalkyl group may be esterified or etherified.
HAS derivatives are described in WO/2004/024776, which is incorporated by
reference in its entirety.

D. Methods of Attachment

[0169] A therapeutic protein may be covalently linked to the
polysaccharide compounds by any of various techniques known to those of
skill in the art. In various aspects of the invention, sialic acid
moieties are bound to a therapeutic protein, e.g., FIX, FVIII, FVIIa or
VWF, for example by the method described in U.S. Pat. No. 4,356,170,
which is herein incorporated by reference.

[0170] Other techniques for coupling PSA to polypeptides are also known
and contemplated by the invention. For example, US Publication No.
2007/0282096 describes conjugating an amine or hydrazide derivative of,
e.g., PSA, to proteins. In addition, US Publication No. 2007/0191597
describes PSA derivatives containing an aldehyde group for reaction with
substrates (e.g., proteins) at the reducing end. These references are
incorporated by reference in their entireties.

[0171] Various methods are disclosed at column 7, line 15, through column
8, line 5 of U.S. Pat. No. 5,846,951 (incorporated by reference in its
entirety). Exemplary techniques include linkage through a peptide bond
between a carboxyl group on one of either the blood coagulation protein
or polysaccharide and an amine group of the blood coagulation protein or
polysaccharide, or an ester linkage between a carboxyl group of the blood
coagulation protein or polysaccharide and a hydroxyl group of the
therapeutic protein or polysaccharide. Another linkage by which the
therapeutic protein is covalently bonded to the polysaccharide compound
is via a Schiff base, between a free amino group on the blood coagulation
protein being reacted with an aldehyde group formed at the non-reducing
end of the polysaccharide by periodate oxidation (Jennings H J and
Lugowski C, J Immunol. 1981; 127:1011-8; Fernandes A I and Gregoriadis G,
Biochim Biophys Acta. 1997; 1341; 26-34). The generated Schiff base is in
one aspect stabilized by specific reduction with NaCNBH3 to form a
secondary amine. An alternative approach is the generation of terminal
free amino groups in the PSA by reductive amination with NH4Cl after
prior oxidation. Bifunctional reagents can be used for linking two amino
or two hydroxyl groups. For example, PSA containing an amino group is
coupled to amino groups of the protein with reagents like BS3
(Bis(sulfosuccinimidyl)suberate/Pierce, Rockford, Ill.). In addition
heterobifunctional cross linking reagents like
Sulfo-EMCS(N-ε-Maleimidocaproyloxy) sulfosuccinimide
ester/Pierce) is used for instance to link amine and thiol groups.

[0172] In another approach, a PSA hydrazide is prepared and coupled to the
carbohydrate moiety of the protein after prior oxidation and generation
of aldehyde functions.

[0173] As described above, a free amine group of the therapeutic protein
reacts with the 1-carboxyl group of the sialic acid residue to form a
peptidyl bond or an ester linkage is formed between the 1-carboxylic acid
group and a hydroxyl or other suitable active group on a blood
coagulation protein. Alternatively, a carboxyl group forms a peptide
linkage with deacetylated 5-amino group, or an aldehyde group of a
molecule of a therapeutic protein forms a Schiff base with the
N-deacetylated 5-amino group of a sialic acid residue.

[0174] Alternatively, the polysaccharide compound is associated in a
non-covalent manner with a therapeutic protein. For example, the
polysaccharide compound and the pharmaceutically active compound are in
one aspect linked via hydrophobic interactions. Other non-covalent
associations include electrostatic interactions, with oppositely charged
ions attracting each other.

[0176] In various embodiments, the therapeutic protein is modified to
introduce glycosylation sites (i.e., sites other than the native
glycosylation sites). Such modification may be accomplished using
standard molecular biological techniques known in the art. Moreover, the
therapeutic protein, prior to conjugation to a water soluble polymer via
one or more carbohydrate moieties, may be glycosylated in vivo or in
vitro. These glycosylated sites can serve as targets for conjugation of
the proteins with water soluble polymers (US Patent Application No.
20090028822, US Patent Application No. 2009/0093399, US Patent
Application No. 2009/0081188, US Patent Application No. 2007/0254836, US
Patent Application No. 2006/0111279, and DeFrees S. et al., Glycobiology,
2006, 16, 9, 833-43). For example, a protein that is not naturally
glycosylated in vivo (e.g., a protein that is not a glycoprotein) may be
modified as described above.

E. Aminooxy Linkage

[0177] In one embodiment of the invention, the reaction of hydroxylamine
or hydroxylamine derivatives with aldehydes (e.g., on a carbohydrate
moiety following oxidation by sodium periodate) to form an oxime group is
applied to the preparation of conjugates of blood coagulation protein.
For example, a glycoprotein (e.g., a therapeutic protein according to the
present invention) is first oxidized with a oxidizing agent such as
sodium periodate (NaIO4) (Rothfus J A et Smith E L., J Biol Chem 1963,
238, 1402-10; and Van Lenten L and Ashwell G., J Biol Chem 1971, 246,
1889-94). The periodate oxidation of glycoproteins is based on the
classical Malaprade reaction described in 1928, the oxidation of vicinal
diols with periodate to form an active aldehyde group (Malaprade L.,
Analytical application, Bull Soc Chim France, 1928, 43, 683-96).
Additional examples for such an oxidizing agent are lead tetraacetate
(Pb(OAc)4), manganese acetate (MnO(Ac)3), cobalt acetate
(Co(OAc)2), thallium acetate (TlOAc), cerium sulfate (Ce(SO4)2)
(U.S. Pat. No. 4,367,309) or potassium perruthenate (KRuO4) (Marko et
al., J Am Chem Soc 1997, 119, 12661-2). By "oxidizing agent" a mild
oxidizing compound which is capable of oxidizing vicinal diols in
carbohydrates, thereby generating active aldehyde groups under
physiological reaction conditions is meant.

[0178] The second step is the coupling of the polymer containing an
aminooxy group to the oxidized carbohydrate moiety to form an oxime
linkage. In one embodiment of the invention, this step can be carried out
in the presence of catalytic amounts of the nucleophilic catalyst aniline
or aniline derivatives (Dirksen A et Dawson P E, Bioconjugate Chem. 2008;
Zeng Y et al., Nature Methods 2009; 6:207-9). The aniline catalysis
dramatically accelerates the oxime ligation allowing the use of very low
concentrations of the reagents. In another embodiment of the invention
the oxime linkage is stabilized by reduction with NaCNBH3 to form an
alkoxyamine linkage (FIG. 2). Additional catalysts are described below.

[0181] As described herein, the conjugation of water soluble polymers to
therapeutic proteins can be catalyzed by aniline. Aniline strongly
catalyzes aqueous reactions of aldehydes and ketones with amines to form
stable imines such as hydrazones and oximes. The following diagram
compares an uncatalyzed versus the aniline-catalyzed oxime ligation
reaction (Kohler J J, ChemBioChem 2009; 10:2147-50):

[0183] In one embodiment of the invention, m-toluidine (aka
meta-toluidine, m-methylaniline, 3-methylaniline, or
3-amino-1-methylbenzene) is used to catalyze the conjugation reactions
described herein. M-toluidine and aniline have similar physical
properties and essentially the same pKa value (m-toluidine: pKa 4.73,
aniline: pKa 4.63).

[0184] The nucleophilic catalysts of the invention are useful for oxime
ligation (e.g, using aminooxy linkage) or hydrazone formation (e.g.,
using hydrazide chemistry). In various embodiments of the invention, the
nucleophilic catalyst is provided in the conjugation reaction at a
concentration of 0.1, 0.2, 0.3, 0.5, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,
45, or 50 mM. In one embodiment, the nucleophilic catalyst is provided
between 1 to 10 mM. In various embodiments of the invention, the pH range
of conjugation reaction is 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5. In one
embodiment, the pH is between 5.5 to 6.5.

Purification of Conjugated Proteins

[0185] In various embodiments, purification of a protein that has been
incubated with an oxidizing agent and/or a therapeutic protein that has
been conjugated with a water soluble polymer according to the present
disclosure, is desired. Numerous purification techniques are known in the
art and include, without limitation, chromatographic methods such as
ion-exchange chromatography, hydrophobic interaction chromatography, size
exclusion chromatography and affinity chromatography or combinations
thereof, filtration methods, and precipitation methods (Guide to Protein
Purification, Meth. Enzymology Vol 463 (edited by Burgess R R and
Deutscher M P), 2nd edition, Academic Press 2009).

[0186] The following examples are not intended to be limiting but only
exemplary of specific embodiments of the invention.

EXAMPLES

Example 1

Preparation of the Homobifunctional Linker
NH2[OCH2CH2]2ONH2

[0187] The homobifunctional linker
NH2[OCH2CH2]2ONH2

##STR00022##

[0188] (3-oxa-pentane-1,5-dioxyamine) containing two active aminooxy
groups was synthesized according to Boturyn et al. (Tetrahedron 1997;
53:5485-92) in a two step organic reaction employing a modified
Gabriel-Synthesis of primary amines (FIG. 3). In the first step, one
molecule of 2,2-chlorodiethylether was reacted with two molecules of
Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in dimethylformamide (DMF).
The desired homobifunctional product was prepared from the resulting
intermediate by hydrazinolysis in ethanol.

Example 2

Preparation of the Homobifunctional Linker
NH2[OCH2CH2]4ONH2

[0189] The homobifunctional linker
NH2[OCH2CH2]4ONH2

##STR00023##

[0190] (3,6,9-trioxa-undecane-1,11-dioxyamine) containing two active
aminooxy groups was synthesized according to Boturyn et al. (Tetrahedron
1997; 53:5485-92) in a two step organic reaction employing a modified
Gabriel-Synthesis of primary amines (FIG. 3). In the first step one
molecule of Bis-(2-(2-chloroethoxy)-ethyl)-ether was reacted with two
molecules of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The
desired homobifunctional product was prepared from the resulting
intermediate by hydrazinolysis in ethanol.

Example 3

Preparation of the Homobifunctional Linker
NH2[OCH2CH2]6ONH2

[0191] The homobifunctional linker
NH2[OCH2CH2]6ONH2

##STR00024##

[0192] (3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine) containing two
active aminooxy groups was synthesized according to Boturyn et al.
(Tetrahedron 1997; 53:5485-92) in a two step organic reaction employing a
modified Gabriel-Synthesis of primary amines. In the first step one
molecule of hexaethylene glycol dichloride was reacted with two molecules
of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desired
homobifunctional product was prepared from the resulting intermediate by
hydrazinolysis in ethanol.

Example 4

Detailed Synthesis of the aminooxy-PSA Reagent

[0193] 3-oxa-pentane-1,5 dioxyamine was synthesized according to Botyryn
et al (Tetrahedron 1997; 53:5485-92) in a two step organic synthesis as
outlined in Example 1.

Step 1:

[0194] To a solution of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide
(59.0 g; 1.00 eq) in 700 ml anhydrous N,N-dimethylformamide anhydrous
K2CO3 (45.51 g; 1.00 eq) and 2,2-dichlorodiethylether (15.84
ml; 0.41 eq) were added. The reaction mixture was stirred for 22 h at
50° C. The mixture was evaporated to dryness under reduced
pressure. The residue was suspended in 2 L dichloromethane and extracted
two times with saturated aqueous NaCl-solution (each 1 L). The
Dichloromethane layer was dried over Na2SO4 and then evaporated
to dryness under reduced pressure and dried in high vacuum to give 64.5 g
of 3-oxapentane-1,5-dioxy-endo-2',3'-dicarboxydiimidenorbornene as a
white-yellow solid (intermediate 1).

Step 2:

[0195] To a solution of intermediate 1 (64.25 g; 1.00 eq) in 800 ml
anhydrous Ethanol, 31.0 ml Hydrazine hydrate (4.26 eq) were added. The
reaction mixture was then refluxed for 2 hrs. The mixture was
concentrated to the half of the starting volume by evaporating the
solvent under reduced pressure. The occurring precipitate was filtered
off. The remaining ethanol layer was evaporated to dryness under reduced
pressure. The residue containing the crude product
3-oxa-pentane-1,5-dioxyamine was dried in vacuum to yield 46.3 g. The
crude product was further purified by column chromatography (Silicagel
60; isocratic elution with Dichloromethane/Methanol mixture, 9/1) to
yield 11.7 g of the pure final product 3-oxa-pentane-1,5-dioxyamine.

Example 5

Preparation of aminooxy-PSA

[0196] 1000 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 16 ml 50 mM phosphate
buffer pH 6.0. Then 170 mg 3-oxa-pentane-1,5-dioxyamine was given to the
reaction mixture. After shaking for 2 hrs at RT 78.5 mg sodium
cyanoborohydride was added and the reaction was performed for 18 hours
over night. The reaction mixture was then subjected to a
ultrafiltration/diafiltration procedure (UF/DF) using a membrane with a 5
kD cut-off made of regenerated cellulose (50 cm2, Millipore).

[0197] 1290 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 25 ml 50 mM phosphate
buffer pH 6.0 (Bufffer A). Then 209 mg 3-oxa-pentane-1,5-dioxyamine was
given to the reaction mixture. After shaking for 1 h at RT 101 mg sodium
cyanoborohydride was added and the reaction was performed for 3 hours.
Then the mixture was then subjected to a weak anion exchange
chromatography step employing a Fractogel EMD DEAE 650-M chromatography
gel (column dimension: XK26/135). The reaction mixture was diluted with
110 ml Buffer A and loaded onto the DEAE column pre-equilibrated with
Buffer A at a flow rate of 1 cm/min. Then the column was washed with 20
CV Buffer B (20 mM Hepes, pH 6.0) to remove free
3-oxa-pentane-1,5-dioxyamine and cyanide at a flow rate of 2 cm/min. The
aminooxy-PSA reagent was then eluted with a step gradient consisting of
67% Buffer B and 43% Buffer C (20 mM Hepes, 1M NaCl, pH 7.5). The eluate
was concentrated by UF/DF using a 5 kD membrane made of polyether sulfone
(50 cm2, Millipore). The final diafiltration step was performed
against Buffer D (20 mM Hepes, 90 mM NaCl, pH 7.4). The preparation was
analytically characterized by measuring total PSA (Resorcinol assay) and
total aminooxy groups (TNBS assay) to determine the degree of
modification. Furthermore the polydispersity as well as free
3-oxa-pentane-1,5-dioxyamine and cyanide was determined.

Example 7

Preparation of aminooxy-PSA without a Reduction Step

[0198] 573 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute
of India (Pune, India) was dissolved in 11.3 ml 50 mM phosphate buffer pH
6.0 (Bufffer A). Then 94 mg 3-oxa-pentane-1,5-dioxyamine was given to the
reaction mixture. After shaking for 5 h at RT the mixture was then
subjected to a weak anion exchange chromatography step employing a
Fractogel EMD DEAE 650-M chromatography gel (column dimension: XK16/105).
The reaction mixture was diluted with 50 ml Buffer A and loaded onto the
DEAE column pre-equilibrated with Buffer A at a flow rate of 1 cm/min.
Then the column was washed with 20 CV Buffer B (20 mM Hepes, pH 6.0) to
remove free 3-oxa-pentane-1,5-dioxyamine and cyanide at a flow rate of 2
cm/min. The aminooxy-PSA reagent was the eluted with a step gradient
consisting of 67% Buffer B and 43% Buffer C (20 mM Hepes, 1 M NaCl, pH
7.5). The eluate was concentrated by UF/DF using a 5 kD membrane made of
polyether sulfone (50 cm2, Millipore). The final diafiltration step
was performed against Buffer D (20 mM Hepes, 90 mM NaCl, pH 7.4). The
preparation was analytically characterized by measuring total PSA
(Resorcinol assay) and total aminooxy groups (TNBS assay) to determine
the degree of modification. Furthermore the polydispersity as well as
free 3-oxa-pentane-1,5-dioxyamine was determined.

Example 8

Preparation of aminooxy-PSA without a Reduction Step in the Presence of
the Nucleophilic Catalyst m-toluidine

[0199] 573 mg of oxidized PSA (MW=20 kD) obtained from the Serum Institute
of India (Pune, India) is dissolved in 9 ml 50 mM phosphate buffer pH 6.0
(Bufffer A). Then 94 mg 3-oxa-pentane-1,5-dioxyamine is given to this
solution. Subsequently 2.3 ml of a 50 mM m-toluidine stock solution are
added to this reaction mixture. After shaking for 2 h at RT the mixture
is then subjected to a weak anion exchange chromatography step employing
a Fractogel EMD DEAE 650-M chromatography gel (column dimension:
XK16/105). The reaction mixture is diluted with 50 ml Buffer A and loaded
onto the DEAE column pre-equilibrated with Buffer A at a flow rate of 1
cm/min. Then the column is washed with 20 CV Buffer B (20 mM Hepes, pH
6.0) to remove free 3-oxa-pentane-1,5-dioxyamine and cyanide at a flow
rate of 2 cm/min. The aminooxy-PSA reagent is the eluted with a step
gradient consisting of 67% Buffer B and 43% Buffer C (20 mM Hepes, 1 M
NaCl, pH 7.5). The eluate is concentrated by UF/DF using a 5 kD membrane
made of polyether sulfone (50 cm2, Millipore). The final
diafiltration step is performed against Buffer D (20 mM Hepes, 90 mM
NaCl, pH 7.4). The preparation is analytically characterized by measuring
total PSA (Resorcinol assay) and total aminooxy groups (TNBS assay) to
determine the degree of modification. Furthermore the polydispersity as
well as free 3-oxa-pentane-1,5-dioxyamine is determined.

Example 9

Preparation of aminooxy-PSA Reagent

[0200] An Aminooxy-PSA reagent was prepared according to the Examples 4-8.
After diafiltration, the product was frozen at -80° C. and
lyophilized. After lyophilization the reagent was dissolved in the
appropriate volume of water and used for preparation of PSA-protein
conjugates via carbohydrate modification.

Example 10

Evaluation of the Efficacy of Different Alternative Nucleophilic Catalysts

[0202] The coupling efficiency was determined by SDS-PAGE using an
Invitrogen X-cell mini system. Samples were spiked with lithium dodecyl
sulfate (LDS) buffer and denatured for 10 min at 70° C. Then the
samples were applied on 3-8% TRIS-acetate gels and ran at 150 V for 60
min. Subsequently the gels were stained with Coomassie.

[0203] In addition the samples were characterized by use of a SEC-HPLC
system using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77).

[0204] 50 μl of samples were injected undiluted and eluted
isocratically with a 0.22 μm filtered solution of 20 mM NaH2PO4, 50 mM
Na2SO4, pH 6.1 at a flow rate of 0.5 ml/min. The elution pattern was
recorded at 280 nm.

[0205] The results are summarized in FIGS. 5A-C and 6 (SDS PAGE) and Table
2 (SEC-HPLC results). The catalytic effect of the different preparations
is demonstrated. It is shown that the use of m-toluidine leads to
equivalent results as obtained with aniline.

[0207] The retentate (8.8 ml), containing oxidized rFIX was mixed with
2.46 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PSA reagent with a MW of 20 kD
(described above) was added to give a 5-fold molar reagent excess. This
mixture was incubated for 2.5 h at RT in the dark under gentle stirring.

[0209] 12.3 mg rFIX is dissolved in L-histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2) to get a final protein
concentration of 1 mg rFIX/ml. A 5 mM aqueous sodium periodate solution
is added to get a final concentration of 100 μM and the reaction
mixture is incubated for 1 hour in the dark at 4° C. under gentle
stirring at pH 6.0 and quenched for 15 min at room temperature by the
addition of an 1 M aqueous L-cysteine solution (or other quenching
reagents) to get a final concentration of 10 mM. The mixture is
subsequently subjected to UF/DF employing Vivaspin 15R 10 kD centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.

[0210] The obtained retentate (8.8 ml), containing oxidized rFIX, is mixed
with an aqueous m-toluidine solution (50 mM) to give a final
concentration of 10 mM and incubated for 30 min at room temperature. Then
aminooxy-PSA reagent with a MW of 20 kD (described above) is added to
give a 5-fold molar reagent excess. This mixture was incubated at pH 6.0
for 2.5 hours at room temperature; 0.5 hours to 18 hours at +4°
C.) in the dark under gentle stirring.

[0214] 25.4 mg rFIX was dissolved in L-histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2) to get a final protein
concentration of 2 mg rFIX/ml. Subsequently an 5 mM aqueous sodium
periodate solution was added within 15 minutes to give a final
concentration of 100 μM, followed by addition of an 50 mM aqueous
m-toluidine solution to get a final concentration of 10 mM within a time
period of 30 minutes. Then the aminooxy-PSA reagent with a MW of 20 kD
(described above) was added to give a 5-fold molar reagent excess. After
correction of the pH to 6.0 the mixture was incubated for 2 h in the dark
at room temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of a 1 M aqueous L-cysteine solution to give
a final concentration of 10 mM.

Polysialylation of rFVIII Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0216] 50 mg rFVIII was transferred into reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain
a protein concentration of 1 mg/ml. To this solution, NaIO4 was
added to give a final concentration of 200 μM. The oxidation was
carried at RT for 30 min in the dark under gentle shaking. Then the
reaction was quenched with cysteine (final concentration: 10 mM) for 60
min at RT. The solution was subjected to an IEX column with a volume of
20 ml (Merck EMD TMAE (M)) which was equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column was equilibrated with 5 CV
Buffer A. Then the oxidized rFVIII was eluted with Buffer B (20 mM Hepes,
5 mM CaCl2, 1M NaCl, pH 7.0). The rFVIII containing fractions were
collected. The protein content was determined (Coomassie, Bradford) and
adjusted to 1 mg/ml with reaction buffer and adjusted to pH 6.0 by
dropwise addition of 0.5 M HCl. Then a 50-fold molar excess of a
aminooxy-PSA reagent with a MW of 20 kD (described above) was added
followed by m-toluidine as a nucleophilic catalyst (final concentration:
10 mM). The coupling reaction was performed for 2 hours in the dark under
gentle shaking at room temperature. The excess of aminooxy-PSA reagent
was removed by means of HIC. The conductivity of the reaction mixture was
raised to 130 mS/cm by adding a buffer containing ammonium acetate (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium
acetate, pH 6.9) and loaded onto a column filled with 80 ml Phenyl
Sepharose FF (GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50
mM Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate was eluted with 50 mM Hepes
buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-rFVIII
containing fractions were collected and subjected to UF/DF by use of a 30
kD membrane made of regenerated cellulose (88 cm2, Millipore). The
preparation was analytically characterized by measuring total protein
(Coomassie, Bradford) and FVIII chromogenic activity. The PSA-rFVIII
conjugate showed a specific activity of >70% in comparison to native
rFVIII was determined.

Method 2:

[0217] 58 mg of recombinant factor VIII (rFVIII) derived from the ADVATE
process in Hepes buffer (50 mM HEPES, ˜350 mM sodium chloride, 5 mM
calcium chloride, 0.1% Polysorbate 80, pH 7.4) is dissolved in reaction
buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a 0.5 N
aqueous HCl solution. Subsequently, a 40 mM aqueous sodium periodate
solution is added within 10 minutes to give a concentration of 200 μM.
The oxidation reaction is carried out for 30+/-5 min at a temperature (T)
of T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0218] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity of
5 ms/cm. This solution is loaded onto the IEX column (bed height: 5.4 cm)
with a column volume of 10 ml using a flow rate of 1.5 cm/min. This
column is subsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8
mixture (w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl2, 1.0
M NaCl, pH 7.0). Then the oxidized rFVIII is eluted with a 50:50 (w/w)
mixture of Buffer A and Buffer B followed by a postelution step with 5 CV
of Buffer B. The elution steps are carried out by use of a flow rate of
1.0 cm/min.

[0219] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to the eluate containing the purified
oxidized rFVIII within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0222] For removal of reaction by-products and anti-chaotropic salt a
second washing step is performed with >5 CV washing buffer 1 (50 mM
Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode
at a flow rate of 2 cm/min. Then elution of purified PSA-rFVIII conjugate
is performed in down flow mode using a step gradient of 40% washing
buffer 2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH
6.9) and 60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5)
at a flow rate of 1 cm/min. The elution of the PSA-rFVIII conjugate is
monitored at UV 280 nm and the eluate containing the conjugate is
collected within <4 CV. The post elution step is performed with >3
CV elution buffer under the same conditions to separate minor and/or non
modified rFVIII from the main product.

[0223] Finally the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 30 kD (88 cm2, Millipore).

[0224] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein, FVIII chromogenic activity and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay). For the conjugate obtained a specific activity
>50% and a PSA degree >5.0 is calculated.

[0227] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent was
added in a 50-fold molar excess to this rFVIII solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) was added within 15 minutes to get a final
concentration of 10 mM. Finally, a 40 mM aqueous sodium periodate
solution was added to give a concentration of 400 μM.

[0228] The reaction mixture was incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction was stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0240] PEGylation of r FVIII Using an aminooxy-PEG Reagent and m-toluidine
as a Nucleophilic Catalyst

Method 1:

[0241] rFVIII is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 14.7 mg
rFVIII is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). Then 296 μl of an aqueous
sodium periodate solution (5 mM) is added and the reaction mixture is
incubated for 1 h in the dark at 4° C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5 μl of a
1 M aqueous cysteine solution. The mixture was subsequently subjected to
UF/DF employing Vivaspin 15R 10 kD centrifugal filtrators to remove
excess periodate, quencher and the byproducts thereof.

[0242] The retentate (10.9 ml), containing oxidized rFVIII, is mixed with
2.94 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PEG reagent with a MW of 20 kD is
added to give a 5-fold molar reagent excess. This mixture was incubated
for 2.5 h at room temperature in the dark under gentle stirring.

[0243] Finally, the PEG-rFVIII conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a 30 kD membrane (50 cm2, Millipore). The preparation is
analytically characterized by measuring total protein (Coomassie,
Bradford) and FVIII chromogenic activity. It is expected that the
PEG-rFVIII conjugate will demonstrate a specific activity of >70% in
comparison to native rFVIII was determined.

Method 2:

[0244] rFVIII is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). A starting
weight or concentration of rFVIII is dissolved in or transferred to a
reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 1.0+/-0.25
mg/ml. Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution. Subsequently a 40 mM aqueous
sodium periodate solution is added within 10 minutes to give a
concentration of 200 μM. The oxidation reaction is carried out for
30+/-5 min at a temperature (T) of T=+22+/-2° C. Then the reaction
is stopped by addition of an aqueous L-cysteine solution (1 M) within 15
minutes at T=+22+/-2° C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.

[0245] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity of 5
ms/cm. This solution is loaded onto the IEX column (bed height: 5.4 cm)
with a column volume of 10 ml using a flow rate of 1.5 cm/min. This
column is subsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8
mixture (w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl2, 1.0 M
NaCl, pH 7.0). Then the oxidized rFVIII is eluted with a 50:50 (w/w)
mixture of Buffer A and Buffer B followed by a postelution step with 5 CV
of Buffer B. The elution steps are carried out by use of a flow rate of
1.0 cm/min.

[0246] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent
is added in a 50-fold molar excess to the eluate containing the purified
oxidized rFVIII within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0254] rFVIII is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of rFVIII is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg rFVIII/ml.
Subsequently, an 5 mM aqueous sodium periodate solution is added within
15 minutes to give a final concentration of 100 μM, followed by
addition of an 50 mM aqueous m-toluidine solution to get a final
concentration of 10 mM within a time period of 30 minutes. Then the
aminooxy-PEG reagent with a MW of 20 kD (described above) is, added to
give a 20-fold molar excess. After correction of the pH to 6.0 the
mixture is incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the addition of a
1 M aqueous L-cysteine solution to give a final concentration of 10 mM.

[0256] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 14

Polysialylation of rFVIIa Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0257] A starting concentration or weight of recombinant factor VIIa
(rFVIIa) is transferred or dissolved in reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous NaOH solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 50 μM. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of T=+22+/-2° C.
Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0258] The oxidized rFVIIa is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity of
5 ms/cm. This solution is loaded onto the IEX column (bed height: 5.4 cm)
with a column volume of 10 ml using a flow rate of 1.5 cm/min. This
column is subsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8
mixture (w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl2, 1.0
M NaCl, pH 7.0). Then the oxidized rFVIIa is eluted with a 50:50 (w/w)
mixture of Buffer A and Buffer B followed by a postelution step with 5 CV
of Buffer B. The elution steps are carried out by use of a flow rate of
1.0 cm/min.

[0259] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to the eluate containing the purified
oxidized rFVIIa within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0262] For removal of reaction by-products and anti-chaotropic salt a
second washing step is performed with >5 CV washing buffer 1 (50 mM
Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode
at a flow rate of 2 cm/min. Then elution of purified rFVIIa conjugate is
performed in down flow mode using a step gradient of 40% washing buffer 2
(50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and
60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow
rate of 1 cm/min. The elution of the PSA-rFVIIa conjugate is monitored at
UV 280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution buffer
under the same conditions to separate minor and/or non modified rFVIIa
from the main product.

[0263] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off (e.g. 10 kD MWCO,
88 cm2, Millipore).

[0264] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

Method 2:

[0265] A starting weight or concentration of rFVIIa is dissolved in or
transferred to a reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0 by drop
wise addition of a 0.5 N aqueous NaOH solution.

[0266] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to this rFVIIa solution within a
maximum time period (t) of 15 minutes under gentle stirring. Then an
aqueous m-toluidine solution (50 mM) is added within 15 minutes to get a
final concentration of 10 mM. Finally a 40 mM aqueous sodium periodate
solution is added to give a concentration of 150 μM.

[0267] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0270] For removal of reaction by-products and anti-chaotropic salt a
second washing step is performed with >5 CV washing buffer 1 (50 mM
Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode
at a flow rate of 2 cm/min. Then elution of purified rFVIIa conjugate is
performed in down flow mode using a step gradient of 40% washing buffer 2
(50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and
60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow
rate of 1 cm/min. The elution of the PSA-rFVIIa conjugate is monitored at
UV 280 nm and the eluate containing the conjugate was collected within
<4 CV. The post elution step is performed with >3 CV elution buffer
under the same conditions to separate minor and/or non modified rFVIII
from the main product.

[0271] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose (Millipore).

[0272] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 15

PEGylation of rFIX Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0273] rFIX is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). A starting
weight or concentration of rFIX is dissolved in or transferred to a
reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 1.0+/-0.25
mg/ml. Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous
sodium periodate solution is added within 10 minutes to give a
concentration of 200 μM. The oxidation reaction is carried out for
30+/-5 min at a temperature (T) of T=+22+/-2° C. Then the reaction
is stopped by addition of an aqueous L-cysteine solution (1 M) within 15
minutes at T=+22+/-2° C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.

[0274] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity of 5
mS/cm. This solution is loaded onto the IEX column (bed height: 5.4 cm)
with a column volume of 10 ml using a flow rate of 1.5 cm/min. This
column is subsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8
mixture (w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl2, 1.0 M
NaCl, pH 7.0). Then the oxidized rFIX is eluted with a 50:50 (w/w)
mixture of Buffer A and Buffer B followed by a postelution step with 5 CV
of Buffer B. The elution steps are carried out by use of a flow rate of
1.0 cm/min.

[0275] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent
is added in a 50-fold molar excess to the eluate containing the purified
oxidized rFIX within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking.

[0278] For removal of reaction by-products and anti-chaotropic salt a
second washing step is performed with >5 CV washing buffer 1 (50 mM
Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode
at a flow rate of 2 cm/min. Then elution of purified rFIX conjugate is
performed in down flow mode using a step gradient of 40% washing buffer 2
(50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and
60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow
rate of 1 cm/min. The elution of the PEG-rFIX conjugate is monitored at
UV 280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution buffer
under the same conditions to separate minor and/or non modified rFIX from
the main product.

[0279] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 10 kD (88 cm2, Millipore).

[0280] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 2:

[0281] rFIX is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of rFIX is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg rFIX/ml. Subsequently,
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of a 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0283] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 16

PEGylation of rFVIIa Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0284] rFVIIa is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). A starting
weight or concentration of rFVIIa is dissolved in or transferred to a
reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 1.0+/-0.25
mg/ml. Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous NaOH solution. Subsequently, a 40 mM aqueous
sodium periodate solution is added within 10 minutes to give a
concentration of 50 μM. The oxidation reaction is carried out for
30+/-5 min at a temperature (T) of T=+22+/-2° C. Then the reaction
is stopped by addition of an aqueous L-cysteine solution (1 M) within 15
minutes at T=+22+/-2° C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.

[0285] The oxidized rFVIIa is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity of 5
mS/cm. This solution is loaded onto the IEX column (bed height: 5.4 cm)
with a column volume of 10 ml using a flow rate of 1.5 cm/min. This
column is subsequently washed (flow rate: 1.5 cm/min) with 5 CV of a 92:8
mixture (w/w) of Buffer A and Buffer B (20 mM Hepes, 5 mM CaCl2, 1.0 M
NaCl, pH 7.0). Then the oxidized rFVIIa is eluted with a 50:50 (w/w)
mixture of Buffer A and Buffer B followed by a postelution step with 5 CV
of Buffer B. The elution steps are carried out by use of a flow rate of
1.0 cm/min.

[0286] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD reagent
is added in a 50-fold molar excess to the eluate containing the purified
oxidized rFVIIa within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0289] For removal of reaction by-products and anti-chaotropic salt a
second washing step is performed with >5 CV washing buffer 1 (50 mM
Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9) in upflow mode
at a flow rate of 2 cm/min. Then elution of purified rFVIIa conjugate is
performed in down flow mode using a step gradient of 40% washing buffer 2
(50 mM Hepes, 1.5 M sodium chloride, 5 mM calcium chloride, pH 6.9) and
60% elution buffer (20 mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow
rate of 1 cm/min. The elution of the PEG-rFVIIa conjugate is monitored at
UV 280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution buffer
under the same conditions to separate minor and/or non modified rFVIIa
from the main product.

[0290] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 10 kD (Millipore).

[0291] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 2:

[0292] rFVIIa is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of rFVIIa is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg rFVIIa/ml. Subsequently
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of a 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0296] The retentate (6.5 ml), containing oxidized rFIX, is mixed with
1.64 ml of an aqueous o-amino benzoic acid (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PSA reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. This
mixture was incubated for 2.5 h at room temperature in the dark under
gentle stirring.

[0297] The further purification of the conjugate is carried out as
described herein.

Method 2:

[0298] A solution of 1 mg rFIX in 0.65 ml sodium phosphate buffer, pH 6.0
containing a 5-fold molar excess of aminooxy-PSA reagent with a MW of 20
kD (described above) was prepared. Then 333 μl of an aqueous o-amino
benzoic acid solution (30 mM) was added as nucleophilic catalyst to give
a final concentration of 10 mM. Subsequently 20 μl of an aqueous
solution of NaIO4 (5 mM) was added yielding in a final concentration
of 100 μM. The coupling process was performed for 2 hours in the dark
under gentle shaking at room temperature and quenched for 15 min at room
temperature by the addition of 1 μl of aqueous cysteine solution (1
M). The further purification of, the conjugate is carried out as
described herein.

Example 18

Polysialylation of EPO Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0299] A starting concentration of erythropoietin (EPO) is transferred
into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein concentration
of 1 mg/ml. To this solution, NaIO4 is added to give a final
concentration of 200 μM. The oxidation is carried at RT for 30 min in
the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at RT.

[0300] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV
Buffer A. The oxidized EPO is eluted with Buffer B (20 mM Hepes, 5 mM
CaCl2, 1M NaCl, pH 7.0). The EPO containing fractions are collected.
The protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5M HCl.

[0301] A 50-fold molar excess of a aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy-PSA reagent is removed by means of HIC. The
conductivity of the reaction mixture is adjusted by adding a buffer
containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally
the PSA-EPO containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (MWCO 10 kD, 50 cm2,
Millipore). The preparation is next analytically characterized by
measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

[0302] In an alternative embodiment, Method 1 is carried out as follows.

[0304] The retentate (approx. 7 ml), containing oxidized EPO, is mixed
with 2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PSA reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at RT in the dark under gentle stirring.

[0305] The free EPO is removed by means of anion exchange chromatography
(AEC). The reaction mixture is diluted with 20 ml Buffer A (50 mM Hepes,
pH 7.5) and loaded onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare,
Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column is
eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 7.5). Free EPO is eluted
by washing the column with 25% Buffer B and the conjugate at 50% Buffer
B. The conductivity of the conjugate containing fractions is subsequently
raised to ˜190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, pH 6.9)
and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GE Healthcare,
Fairfield, Conn.) pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl,
pH 6.9). Free PSA-reagent is washed out within 5 CV Buffer D.
Subsequently, the conjugate is eluted with 100% Buffer E (50 mM Hepes, pH
7.4). The conjugate containing fractions are concentrated by UF/DF using
a 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
histidine buffer, pH 7.2 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art. For the
PSA-EPO conjugate a specific activity of >50% in comparison to native
EPO is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC system
equipped with a Shodex KW 803 column under conditions as previously
described (Kolarich et al, Transfusion 2006; 46:1959-77). It is shown
that the preparation contains no free EPO.

Method 2:

[0306] EPO is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0307] The oxidized EPO is further purified by ion exchange
chromatography. The oxidized EPO containing fractions of the eluate are
collected and used for the conjugation reaction.

[0308] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized EPO
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0309] The obtained PSA-EPO conjugate is further purified by ion exchange
chromatography. The PSA-EPO conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0310] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0314] EPO is dissolved in or transferred to a reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0315] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this EPO solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0316] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0317] The obtained PSA-EPO conjugate is purified by ion-exchange
chromatography. The PSA-EPO containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (MWCO 10 kD, 88 cm2, Millipore).

[0318] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 19

Polysialylation of Ang-2 Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0319] A starting concentration of angiopoietin-2 (Ang-2) is transferred
into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein concentration
of 1 mg/ml. To this solution, NaIO4 is added to give a final
concentration of 200 μM. The oxidation is carried at RT for 30 min in
the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at RT.

[0320] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts, or, in the alternative, subjected to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A
(20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV
Buffer A. The oxidized Ang-2 is eluted with Buffer B (20 mM Hepes, 5 mM
CaCl2, 1 M NaCl, pH 7.0). The Ang-2 containing fractions are collected.
The protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5 M HCl.

[0321] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-Ang-2-containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0322] In an alternative embodiment, Method 1 is carried out as follows.
Angiopoietin-2 (Ang-2) is transferred into a reaction buffer (e.g., 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. To this solution, NaIO4 is
added to give a final concentration of 200 μM. The oxidation is
carried at RT for 30 min in the dark under gentle shaking. The reaction
is then quenched with cysteine (final concentration: 10 mM) for 60 min at
R.T.

[0323] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0324] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-Ang-2 conjugate-containing fractions of the
eluate are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.

Method 2:

[0325] Ang-2 is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0326] The oxidized Ang-2 is further purified by ion exchange
chromatography. The oxidized Ang-2 containing fractions of the eluate are
collected and used for the conjugation reaction.

[0327] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized Ang-2
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+/-2° C. under gentle shaking (protein concentration: 1 mg/ml).

[0328] The obtained PSA-Ang-2 conjugate is further purified by
ion-exchange chromatography.

[0329] The PSA-Ang-2 conjugate containing fractions are collected and
concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0330] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0332] In an alternative embodiment, Method 3 is carried out as follows.
Angiopoietin-2 (Ang-2) is transferred into reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of a
PSA aminooxy reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. PSA Ang-2-containing fractions
of the eluate are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0333] Ang-2 is dissolved in or transferred to a reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0334] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this Ang-2 solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0335] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0336] The obtained PSA-Ang-2 conjugate is purified by ion-exchange
chromatography. The PSA-Ang-2 containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0337] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 20

Polysialylation of VEGF Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0338] A starting concentration of vascular endothelial growth factor
(VEGF) is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added to
give a final concentration of 200 μM. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at RT.

[0339] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized VEGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1
M NaCl, pH 7.0). The VEGF containing fractions are collected. The protein
content is determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M NaOH.

[0340] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-VEGF-containing fractions are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The preparation
is next analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the art.

[0341] In an alternative embodiment, Method 1 is carried out as follows.
Vascular endothelial growth factor (VEGF) is transferred into a reaction
buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200 μM. The
oxidation is carried at RT for 30 min in the dark under gentle shaking.
The reaction is then quenched with cysteine (final concentration: 10 mM)
for 60 min at RT.

[0342] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0343] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-VEGF-containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

Method 2:

[0344] VEGF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 μM. The oxidation
reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0345] The oxidized VEGF is further purified by ion exchange
chromatography. The oxidized VEGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0346] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized VEGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0347] The obtained PSA-VEGF conjugate is further purified by ion exchange
chromatography. The PSA-VEGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0348] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0350] In an alternative embodiment, Method 3 is carried out as follows.
Vascular endothelial growth factor (VEGF) is transferred into reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (10 mM final concentration) and NaIO4 (final concentration: 400
μM). The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10 mM)
and the conjugate is purified by ion exchange chromatography. The
PSA-VEGF containing fractions of the eluate are collected and subjected
to UF/DF by use of a membrane made of regenerated cellulose (Millipore).
The preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.

Method 4:

[0351] VEGF is dissolved in or transferred to a reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0352] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this VEGF solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0353] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0354] The obtained VEGF-conjugate is purified by ion-exchange
chromatography. The PSA-VEGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0355] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 21

Polysialylation of EGF Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0356] A starting concentration of epidermal growth factor (EGF) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give a
final concentration of 200 μM. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at R.T.

[0357] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized EGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1M
NaCl, pH 7.0). The EGF containing fractions are collected. The protein
content is determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M HCl.

[0358] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-EGF containing fractions are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The preparation
is next analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the art.

[0359] In an alternative embodiment, Method 1 is carried out as follows.
Epidermal growth factor (EGF) is transferred into a reaction buffer
(e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH
6.0) and diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200 μM. The
oxidation is carried at RT for 30 min in the dark under gentle shaking.
The reaction is then quenched with cysteine (final concentration: 10 mM)
for 60 min at R.T.

[0360] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0361] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-EGF containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

Method 2:

[0362] EGF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0363] The oxidized EGF is further purified by ion exchange
chromatography. The oxidized EGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0364] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized EGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0365] The obtained PSA-EGF conjugate is further purified by ion exchange
chromatography. The PSA-EGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0366] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0368] In an alternative embodiment, Method 3 is carried out as follows.
Epidermal growth factor (EGF) is transferred into reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. A 50-fold molar
excess of a PSA aminooxy reagent with a MW of 20 kD (described above) is
added followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. The conjugate containing
fractions of the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0369] EGF is dissolved in or transferred to a reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0370] Subsequently the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this EGF-solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0371] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0372] The obtained EGF-conjugate is purified by ion-exchange
chromatography. The PSA-EGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0373] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 22

Polysialylation of NGF Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0374] A starting concentration of nerve growth factor (NGF) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give a
final concentration of 200 μM. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at RT.

[0375] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized NGF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1M
NaCl, pH 7.0). The NGF containing fractions are collected. The protein
content is determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M HCl.

[0376] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-NGF containing fractions are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The preparation
is next analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the art.

[0377] In an alternative embodiment, Method 1 is carried out as follows.
Nerve growth factor (NGF) is transferred into a reaction buffer (e.g., 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this solution,
NaIO4 is added to give a final concentration of 200 μM. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10 mM) for
60 min at RT.

[0378] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0379] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-NGF containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

Method 2:

[0380] NGF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0381] The oxidized NGF is further purified by ion exchange
chromatography. The oxidized NGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0382] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized NGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0383] The obtained PSA-NGF conjugate is further purified by ion exchange
chromatography. The PSA-NGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0384] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0386] In an alternative embodiment, Method 3 is carried out as follows.
Nerve growth factor (NGF) is transferred into reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. Then the PSA-NGF containing
fractions of the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0387] NGF is dissolved in or transferred to a reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0388] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this NGF-solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0389] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0390] The obtained NGF-conjugate is purified by ion-exchange
chromatography. The PSA-NGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0391] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 23

Polysialylation of HGH Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0392] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0393] A starting concentration of human growth hormone (HGH) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give a
final concentration of 200 μM. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at RT.

[0394] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized HGH is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M
NaCl, pH 7.0). The HGH containing fractions are collected. The protein
content is determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5 M HCl.

[0395] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-HGH containing fractions are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The preparation
is next analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the art.

[0396] In an alternative embodiment, Method 1 is carried out as follows.
As described herein, the amino acid sequence of human growth hormone
(HGH) is first modified to incorporate at least one glycosylation site.
Following purification, HGH is glycosylated in vitro according to methods
known in the art. HGH is transferred into a reaction buffer (e.g., 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. To this solution, NaIO4 is
added to give a final concentration of 200 μM. The oxidation is
carried at RT for 30 min in the dark under gentle shaking. The reaction
is then quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.

[0397] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0398] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-HGH containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

Method 2:

[0399] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0400] HGH is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0401] The oxidized HGH is further purified by ion exchange
chromatography. The oxidized HGH containing fractions of the eluate are
collected and used for the conjugation reaction.

[0402] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized HGH
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0403] The obtained PSA-HGH conjugate is further purified by ion exchange
chromatography. The PSA-HGH conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0404] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

Method 3:

[0405] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0407] In an alternative embodiment, Method 3 is carried out as follows.
As described herein, the amino acid sequence of human growth hormone
(HGH) is first modified to incorporate at least one glycosylation site.
Following purification, HGH is glycosylated in vitro according to methods
known in the art. HGH is transferred into reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. A 50 fold molar excess of
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. Then the PSA-HGH-containing
fractions of the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0408] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0409] HGH is dissolved in or transferred to a reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0410] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this HGH-solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0411] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0412] The obtained HGH-conjugate is purified by ion-exchange
chromatography. The PSA-HGH containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0413] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 24

Polysialylation of TNF-alpha Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

[0414] A starting concentration of tumor necrosis factor-alpha (TNF-alpha)
is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give a
final concentration of 200 μM. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched with
cysteine (final concentration: 10 mM) for 60 min at RT.

[0415] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized TNF-alpha is eluted with Buffer B (20 mM Hepes, 5 mM
CaCl2, 1M NaCl, pH 7.0). The TNF-alpha containing fractions are
collected. The protein content is determined (Coomassie, Bradford) and
adjusted to 1 mg/ml with reaction buffer and adjusted to pH 6.0 by
dropwise addition of 0.5M HCl.

[0416] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-TNF-alpha-containing fractions are collected and subjected to UF/DF
by use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0417] In an alternative embodiment, Method 1 is carried out as follows.
Tumor necrosis factor-alpha (TNF-alpha) is transferred into a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200 μM. The
oxidation is carried at RT for 30 min in the dark under gentle shaking.
The reaction is then quenched with cysteine (final concentration: 10 mM)
for 60 min at RT.

[0418] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent with a
MW of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. The excess of aminooxy reagent is removed by means of ion
exchange chromatography. The PSA-TNF-alpha containing fractions of the
eluate are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.

Method 2:

[0419] TNF-alpha is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0420] The oxidized TNF-alpha is further purified by ion exchange
chromatography. The oxidized TNF-alpha containing fractions of the eluate
are collected and used for the conjugation reaction.

[0421] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
TNF-alpha within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0422] The obtained PSA-TNF-alpha conjugate is further purified by ion
exchange chromatography. The PSA-TNF-alpha conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0423] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0425] In an alternative embodiment, Method 3 is carried out as follows.
Tumor necrosis factor-alpha (TNF-alpha) is transferred into reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (10 mM final concentration) and NaIO4 (final concentration: 400
μM). The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10 mM)
and the conjugate is purified by ion exchange chromatography. The
PSA-TNF-alpha containing fractions of the eluate are collected and
subjected to UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is analytically characterized by measuring
total protein (Bradford) and biological activity according to methods
known in the art.

Method 4:

[0426] TNF-alpha is dissolved in or transferred to a reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0427] Subsequently the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this TNF-alpha-solution within a
maximum time period (t) of 15 minutes under gentle stirring. Then an
aqueous m-toluidine solution (50 mM) is added within 15 minutes to get a
final concentration of 10 mM. Finally a 40 mM aqueous sodium periodate
solution is added to give a concentration of 400 μM.

[0428] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0429] The obtained TNF-alpha conjugate is purified by ion-exchange
chromatography. The PSA-TNF-alpha containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0430] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 25

Polysialylation of Insulin Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0431] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art. A starting concentration of insulin is transferred into a
reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of 1
mg/ml. To this solution, NaIO4 is added to give a final concentration of
200 μM. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.

[0432] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized insulin is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2,
1 M NaCl, pH 7.0). The insulin containing fractions are collected. The
protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5 M HCl.

[0433] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-insulin containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0434] In an alternative embodiment, Method 1 is carried out as follows.
As described herein, the amino acid sequence of insulin is first modified
to incorporate at least one glycosylation site. Following purification,
insulin is glycosylated in vitro according to methods known in the art.
Insulin is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added to
give a final concentration of 200 μM. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at RT.

[0435] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0436] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion exchange
chromatography. The PSA-insulin containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological activity
according to methods known in the art.

Method 2:

[0437] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0438] Insulin is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0439] The oxidized insulin is further purified by ion exchange
chromatography. The oxidized insulin containing fractions of the eluate
are collected and used for the conjugation reaction.

[0440] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
insulin within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0441] The obtained PSA-insulin conjugate is further purified by ion
exchange chromatography. The PSA-insulin conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0442] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

Method 3:

[0443] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0445] In an alternative embodiment, Method 3 is carried out as follows.
As described herein, the amino acid sequence of insulin is first modified
to incorporate at least one glycosylation site. Following purification,
insulin is glycosylated in vitro according to methods known in the art.

[0446] Insulin is transferred into reaction buffer (e.g. 50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain
a protein concentration of 1 mg/ml. A 50-fold molar excess of
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. PSA-insulin containing fractions
of the eluate are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0447] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0448] Insulin is dissolved in or transferred to a reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0449] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this insulin-solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400 μM.

[0450] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0451] The obtained insulin conjugate is purified by ion-exchange
chromatography. The PSA-insulin containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0452] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 26

Polysialylation of Interferon-alpha Using aminooxy-PSA and m-toluidine as
a Nucleophilic Catalyst

Method 1:

[0453] A starting concentration of interferon-alpha is transferred into a
reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of 1
mg/ml. To this solution, NaIO4 is added to give a final concentration of
200 μM. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.

[0454] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized interferon-alpha is eluted with Buffer B (20 mM Hepes, 5
mM CaCl2, 1M NaCl, pH 7.0). The interferon-alpha containing fractions are
collected. The protein content is determined (Coomassie, Bradford) and
adjusted to 1 mg/ml with reaction buffer and adjusted to pH 6.0 by
dropwise addition of 0.5 M HCl.

[0455] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-interferon-alpha containing fractions are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0456] In an alternative embodiment, Method 1 is carried out as follows.
Interferon-alpha is transferred into a reaction buffer (e.g. 50 mM Hepes,
350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4 is
added to give a final concentration of 200 μM. The oxidation is
carried at RT for 30 min in the dark under gentle shaking. The reaction
is then quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.

[0457] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.

[0458] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of ion-exchange
chromatography. The PSA-interferon-alpha containing fractions of the
eluate are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.

Method 2:

[0459] Interferon-alpha is transferred or dissolved in reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of
the solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous
HCl solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0460] The oxidized interferon-alpha is further purified by ion exchange
chromatography. The oxidized interferon-alpha containing fractions of the
eluate are collected and used for the conjugation reaction.

[0461] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
interferon-gamma within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. at pH 6.0 in the dark at a
temperature (T) of T=+22+/-2° C. under gentle shaking (protein
concentration: 1 mg/ml).

[0462] The obtained PSA-interferon-alpha conjugate is further purified by
ion exchange chromatography. The PSA-interferon-alpha conjugate
containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off (Millipore).

[0464] In an alternative embodiment, Method 3 is carried out as follows.
Interferon-alpha is transferred into reaction buffer (e.g. 50 mM Hepes,
350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. A 50-fold molar excess of
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 μM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking at
room temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate is
purified by ion exchange chromatography. The PSA-interferon-alpha
containing fractions of the eluate are collected and subjected to UF/DF
by use of a membrane made of regenerated cellulose (Millipore). The
preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.

Method 4:

[0465] Interferon-alpha is dissolved in or transferred to a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml. Then
the pH of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution.

[0466] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this interferon-alpha solution within
a maximum time period (t) of 15 minutes under gentle stirring. Then an
aqueous m-toluidine solution (50 mM) is added within 15 minutes to get a
final concentration of 10 mM. Finally, a 40 mM aqueous sodium periodate
solution is added to give a concentration of 400 μM.

[0467] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0468] The obtained interferon-alpha conjugate is purified by ion-exchange
chromatography. The PSA-interferon-alpha containing fractions of the
eluate are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).

[0469] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 27

Polysialylation of Interferon-gamma Using aminooxy-PSA and m-toluidine as
a Nucleophilic Catalyst

[0471] The retentate (approx. 7 ml), containing oxidized interferon-gamma,
is mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PSA reagent with
a MW of 20 kD (described above) is added to give a 5-fold molar reagent
excess. This mixture is incubated for 2.5 h at RT in the dark under
gentle stirring.

[0477] Interferon-gamma is dissolved in or transferred to a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml. Then
the pH of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution.

[0478] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this interferon-gamma solution within
a maximum time period (t) of 15 minutes under gentle stirring. Then an
aqueous m-toluidine solution (50 mM) is added within 15 minutes to get a
final concentration of 10 mM. Finally, a 40 mM aqueous sodium periodate
solution is added to give a concentration of 400 μM.

[0479] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0480] The obtained interferon-gamma conjugate is purified by ion-exchange
chromatography. The PSA-interferon-gamma containing fractions of the
eluate are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).

[0481] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 28

Polysialylation of G-CSF Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0482] A starting concentration of granulocyte-colony stimulating factor
(G-CSF) is transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added to
give a final concentration of 200 μM. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at RT.

[0483] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized G-CSF is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1
M NaCl, pH 7.0). The G-CSF containing fractions are collected. The
protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5 M HCl.

[0484] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently, the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-G-CSF-containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0485] In an alternative embodiment, Method 1 is carried out as follows.
Granulocyte-colony stimulating factor (G-CSF) is transferred into a
reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of 1
mg/ml. To this solution, NaIO4 is added to give a final concentration of
200 μM. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.

[0486] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent with a
MW of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. The excess of aminooxy reagent is removed by means of ion
exchange chromatography. The PSA-G-CSF containing fractions of the eluate
are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.

Method 2:

[0487] G-CSF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0488] The oxidized G-CSF is further purified by ion exchange
chromatography. The oxidized G-CSF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0489] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized G-CSF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0490] The obtained PSA-G-CSF conjugate is further purified by ion
exchange chromatography. The PSA-G-CSF conjugate containing fractions are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0491] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0493] In an alternative embodiment, Method 3 is carried out as follows.
Granulocyte-colony stimulating factor (G-CSF) is transferred into
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of 1
mg/ml. A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (10 mM final concentration) and NaIO4 (final concentration: 400
μM). The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10 mM)
and the conjugate is purified by ion exchange chromatography. The
PSA-G-CSF containing fractions of the eluate are collected and subjected
to UF/DF by use of a membrane made of regenerated cellulose (Millipore).
The preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.

Method 4:

[0494] G-CSF is dissolved in or transferred to a reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0495] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this G-CSF solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally, a 40 mM aqueous sodium periodate
solution is added to give a concentration of 400 μM.

[0496] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0497] The obtained G-CSF conjugate is purified by ion-exchange
chromatography. The PSA-G-CSF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0498] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 29

Polysialylation of Humira Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0499] A starting concentration of Humira is transferred into a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200 μM. The
oxidation is carried at RT for 30 min in the dark under gentle shaking.
The reaction is then quenched with cysteine (final concentration: 10 mM)
for 60 min at RT.

[0500] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized Humira is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2,
1M NaCl, pH 7.0). The Humira containing fractions are collected. The
protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5M HCl.

[0501] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-Humira containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0502] In an alternative embodiment, Method 1 is carried out as follows.
Humira is transferred into a reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added to
give a final concentration of 200 μM. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at RT.

[0503] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent with a
MW of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. The excess of aminooxy reagent is removed by means of ion
exchange chromatography The PSA-Humira containing fractions of the elutae
are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.

Method 2:

[0504] Humira is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0505] The oxidized Humira is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the eluate
are collected and used for the conjugation reaction.

[0506] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
Humira within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0507] The obtained PSA-Humira conjugate is further purified by ion
exchange chromatography. The PSA-Humira conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0508] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0510] In an alternative embodiment, Method 3 is carried out as follows.
Humira is transferred into reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration) and
NaIO4 (final concentration: 400 μM). The coupling reaction is
performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine for 60
min at RT (cysteine concentration: 10 mM) and the conjugate is purified
by ion exchange chromatography. The PSA-Humira containing fractions of
the eluate are collected and subjected to UF/DF by use of a membrane made
of regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.

Method 4:

[0511] Humira is dissolved in or transferred to a reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0512] Subsequently, the aminooxy-polysialic acid (PSA-ONH2) reagent is
added in a 50-fold molar excess to this Humira solution within a maximum
time period (t) of 15 minutes under gentle stirring. Then an aqueous
m-toluidine solution (50 mM) is added within 15 minutes to get a final
concentration of 10 mM. Finally a 40 mM aqueous sodium periodate solution
is added to give a concentration of 400

[0513] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0514] The obtained Humira-conjugate is purified by ion-exchange
chromatography. The PSA-Humira containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0515] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 30

Polysialylation of Prolia Using aminooxy-PSA and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0516] A starting concentration of Prolia is transferred into a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200 μM. The
oxidation is carried at RT for 30 min in the dark under gentle shaking.
The reaction is then quenched with cysteine (final concentration: 10 mM)
for 60 min at RT.

[0517] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a volume
of 20 ml (Merck EMD TMAE (M)) which is equilibrated with Buffer A (20 mM
Hepes, 5 mM CaCl2, pH 7.0). The column is equilibrated with 5 CV Buffer
A. The oxidized Prolia is eluted with Buffer B (20 mM Hepes, 5 mM CaCl2,
1M NaCl, pH 7.0). The Prolia containing fractions are collected. The
protein content is determined (Coomassie, Bradford) and adjusted to 1
mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise addition of
0.5 M HCl.

[0518] A 50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a nucleophilic
catalyst (final concentration: 10 mM). The coupling reaction is performed
for 2 hours in the dark under gentle shaking at room temperature. The
excess of aminooxy reagent is removed by means of HIC. The conductivity
of the reaction mixture is adjusted by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column filled
with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-Prolia containing fractions are collected and subjected to UF/DF by
use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0519] In an alternative embodiment, Method 1 is carried out as follows.
10 mg Prolia is dissolved in 5 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl). 100 μl of an aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 50 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin 15R 10 kD centrifugal filtrators to remove excess
periodate, quencher and the byproducts thereof.

[0520] The retentate (approx. 7 ml), containing oxidized Prolia, is mixed
with 2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PSA reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at RT in the dark under gentle stirring.

[0522] Prolia is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0523] The oxidized Prolia is further purified by ion exchange
chromatography. The oxidized Prolia containing fractions of the eluate
are collected and used for the conjugation reaction.

[0524] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
Prolia within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. at pH 6.0 in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking (protein concentration: 1
mg/ml).

[0525] The obtained Prolia conjugate is further purified by ion exchange
chromatography. The Prolia conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0526] The conjugate prepared by use of this procedure is analytically
characterized by measuring total protein, biological activity, and
determination of the polysialyation degree by measuring the PSA content
(resorcinol assay).

[0530] Prolia is dissolved in or transferred to a reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.

[0531] Subsequently the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to this Prolia-solution within a
maximum time period (t) of 15 minutes under gentle stirring. Then an
aqueous m-toluidine solution (50 mM) is added within 15 minutes to get a
final concentration of 10 mM. Finally a 40 mM aqueous sodium periodate
solution is added to give a concentration of 400 μM.

[0532] The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2° C. under gentle shaking. Then the
reaction is stopped by the addition of an aqueous L-cysteine solution (1
M) to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0533] The obtained Prolia conjugate is purified by ion-exchange
chromatography. The PSA-Prolia containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).

[0534] The conjugates prepared by use of this procedure are analytically
characterized by measuring total protein, biological activity according
to methods known in the art, and determination of the polysialyation
degree by measuring the PSA content (resorcinol assay).

Example 31

Polysialylation of Other Therapeutic Proteins

[0535] Polysialylation reactions performed in the presence of alternative
nucleophilic catalysts like m-toluidine or o-aminobenzoic acid as
described herein may be extended to other therapeutic proteins. For
example, in various aspects of the invention, the above polysialylation
or PEGylation reactions as described herein with PSA aminooxy or PEG
aminooxy reagents is repeated with therapeutic proteins such as those
proteins described herein.

Example 32

PEGylation of EPO Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0536] Erythropoietin (EPO) is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this type
of reagent is the Sunbright® CA series from NOF (NOF Corp., Tokyo,
Japan). EPO is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 7.5 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0537] The retentate containing oxidized EPO is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0538] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography (e.g. on Q Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0539] In an alternative embodiment, Method 1 is carried out as follows.
EPO is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg EPO is
dissolved in 5 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl). 100 μl of an aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 50 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0540] The retentate (approx. 7 ml), containing oxidized EPO, is mixed
with 2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at RT in the dark under gentle stirring.

[0541] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. The reaction mixture is diluted with 20
ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with
Buffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 M NaCl,
pH 7.5). Free EPO is eluted by washing the column with 25% Buffer B and
the conjugate at 50% Buffer B. The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against histidine buffer, pH 7.2 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total protein
(Bradford) and biological activity biological activity according to
methods known in the art. For the PEG-EPO conjugate a specific activity
of >50% in comparison to native EPO is determined. The conjugate is
additionally analytically characterized by Size Exclusion HPLC using a
Agilent 1200 HPLC system equipped with a Shodex KW 803 column under
conditions as previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free EPO.

Method 2:

[0542] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).

[0543] EPO is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 μM. The oxidation
reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0544] The oxidized EPO is further purified by ion exchange
chromatography. The oxidized EPO containing fractions of the eluate are
collected and used for the conjugation reaction.

[0545] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized EPO
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0546] The obtained PEG-EPO conjugate is further purified by ion exchange
chromatography. The PEG-EPO conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0547] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0548] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EPO is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0549] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0550] In an alternative embodiment, Method 3 is carried out as follows.
EPO is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg EPO is
dissolved in ˜8 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150
mM NaCl). 200 μl of an aqueous sodium periodate solution (5 mM) and 2
ml of an aqueous m-toluidine solution (50 mM) are then added.
Subsequently, the aminooxy-PEG reagent with a MW of 20 kD (described
above) is added to give a 5-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle stirring
and quenched for 15 min at room temperature by the addition of 100 μl
of 1 M aqueous cysteine solution.

[0551] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. The reaction mixture is diluted with 20
ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with
Buffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 M NaCl,
pH 7.5). Free EPO is eluted by washing the column with 25% Buffer B and
the conjugate at 50% Buffer B. The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against histidine buffer, pH 7.2 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.
For the PEG-EPO conjugate a specific activity of >50% in comparison to
native EPO is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC system
equipped with a Shodex KW 803 column under conditions as previously
described (Kolarich et al, Transfusion 2006; 46:1959-77). It is shown
that the preparation contains no free EPO.

Method 4:

[0552] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of EPO is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg EPO/ml. Subsequently an
5 mM aqueous sodium periodate solution is added within 15 minutes to give
a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of a 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0553] The PEG-EPO conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration
step is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH
7.5).

[0554] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 33

PEGylation of Ang-2 Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0555] Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0556] The retentate containing oxidized Ang-2 is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0557] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography (e.g. on Q Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0558] In an alternative embodiment, Method 1 is carried out as follows.
Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0559] The retentate containing oxidized Ang-2 is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0560] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography. The conjugate containing fraction of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0561] Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).

[0562] Ang-2 is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl
solution. Subsequently a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 μM. The oxidation
reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0563] The oxidized Ang-2 is further purified by ion exchange
chromatography. The oxidized Ang-2 containing fractions of the eluate are
collected and used for the conjugation reaction.

[0564] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized Ang-2
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0565] The obtained PEG-Ang-2 conjugate is further purified by ion
exchange chromatography. The PEG-Ang-2 conjugate containing fractions are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0566] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0567] Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min of room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0568] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0569] In an alternative embodiment, Method 3 is carried out as follows.
Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Ang-2 is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0570] Finally the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.

Method 4:

[0571] Ang-2 is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of Ang-2 is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg Ang-2/ml. Subsequently
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0572] The PEG-Ang-2 conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0573] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

[0574] Subsequently, the free Ang-2 is removed by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF.

Example 34

PEGylation of VEGF Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0575] VEGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0576] The retentate containing oxidized VEGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0577] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0578] In an alternative embodiment, Method 1 is carried out as follows.
VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0579] The retentate containing oxidized VEGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5'-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0580] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0581] VEGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 200 μM. The oxidation reaction
is carried out for 30+/-5 min at a temperature (T) of T=+22+/-2°
C. Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0582] The oxidized VEGF is further purified by ion exchange
chromatography. The oxidized VEGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0583] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized VEGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0584] The obtained PEG-VEGF conjugate is further purified by ion exchange
chromatography. The PEG-VEGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0585] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0586] VEGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0587] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0588] In an alternative embodiment, Method 3 is carried out as follows.
VEGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). VEGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0589] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography. The conjugate fractions of the eluate are collected and
then subjected to UF/DF. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

Method 4:

[0590] VEGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of VEGF is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg VEGF/ml. Subsequently,
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0591] The PEG-VEGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0592] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 35

PEGylation of EGF Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0593] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0594] The retentate containing oxidized EGF is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0595] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0596] In an alternative embodiment, Method 1 is carried out as follows.
EGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0597] The retentate containing oxidized EGF is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0598] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0599] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EGF is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 200 μM. The oxidation reaction
is carried out for 30+/-5 min at a temperature (T) of T=+22+/-2°
C. Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0600] The oxidized EGF is further purified by ion exchange
chromatography. The oxidized EGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0601] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized NGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0602] The obtained PEG-EGF conjugate is further purified by ion exchange
chromatography. The PEG-EGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0603] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0604] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0605] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0606] In an alternative embodiment, Method 3 is carried out as follows.
EGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0607] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.

Method 4:

[0608] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of EGF is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg EGF/ml. Subsequently an
5 mM aqueous sodium periodate solution is added within 15 minutes to give
a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0609] The PEG-EGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0610] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 36

PEGylation of NGF Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0611] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). NGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0612] The retentate containing oxidized NGF is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0613] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM. Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0614] In an alternative embodiment, Method 1 is carried out as follows.
NGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). NGF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0615] The retentate containing oxidized NGF is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0616] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0617] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). NGF is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently a 40 mM aqueous sodium periodate solution is added within 10
minutes to give a concentration of 200 μM. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of T=+22+/-2° C.
Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0618] The oxidized NGF is further purified by ion exchange
chromatography. The oxidized NGF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0619] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized NGF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0620] The obtained PEG-NGF conjugate is further purified by ion exchange
chromatography. The PEG-NGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0621] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0622] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). NGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0623] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0624] In an alternative embodiment, Method 3 is carried out as follows.
NGF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). NGF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0625] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected and then
subjected to UF/DF. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

Method 4:

[0626] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of NGF is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg NGF/ml. Subsequently an
5 mM aqueous sodium periodate solution is added within 15 minutes to give
a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0627] The PEG-NGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0628] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 37

PEGylation of HGH Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0629] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0630] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). HGH is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0631] The retentate containing oxidized HGH is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0632] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0633] In n alternative embodiment, Method 1 is carried out as follows. As
described herein, the amino acid sequence of human growth hormone (HGH)
is first modified to incorporate at least one glycosylation site.
Following purification, HGH is glycosylated in vitro according to methods
known in the art.

[0634] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). HGH is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0635] The retentate containing oxidized HGH is next mixed with an aqueous
m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0636] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0637] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0638] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). HGH is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 200 μM. The oxidation reaction
is carried out for 30+/-5 min at a temperature (T) of T=+22+/-2°
C. Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0639] The oxidized HGH is further purified by ion exchange
chromatography. The oxidized HGH containing fractions of the eluate are
collected and used for the conjugation reaction.

[0640] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized HGH
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0641] The obtained PEG-HGH conjugate is further purified by ion exchange
chromatography. The PEG-NGF conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane made of
regenerated cellulose with an appropriate molecular weight cut off
(Millipore).

[0642] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0643] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0644] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). HGH is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0645] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0646] In an alternative embodiment, Method 3 is carried out as follows.
As described herein, the amino acid sequence of human growth hormone
(HGH) is first modified to incorporate at least one glycosylation site.
Following purification, HGH is glycosylated in vitro according to methods
known in the art. HGH is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). HGH
is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0647] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected and then
subjected to UF/DF. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

Method 4:

[0648] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one glycosylation
site. Following purification, HGH is glycosylated in vitro according to
methods known in the art.

[0649] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of HGH is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg HGH/ml. Subsequently an
5 mM aqueous sodium periodate solution is added within 15 minutes to give
a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of a 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0650] The PEG-HGH conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0651] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 38

PEGylation of TNF-alpha Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0652] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0653] The retentate containing oxidized TNF-alpha is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0654] Finally, the PEG-TNF-alpha conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0655] In an alternative embodiment, Method 1 is carried out as follows.
TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0656] The retentate containing oxidized TNF-alpha is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0657] Finally, the PEG-TNF-alpha conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0658] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently a 40 mM aqueous sodium periodate solution is added within 10
minutes to give a concentration of 200 μM. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of T=+22+/-2° C.
Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0659] The oxidized TNF-alpha is further purified by ion exchange
chromatography. The oxidized TNF-alpha containing fractions of the eluate
are collected and used for the conjugation reaction.

[0660] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized TNF
alpha within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking.

[0661] The obtained PEG-TNF-alpha conjugate is further purified by ion
exchange chromatography. The PEG-TNF-alpha conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0662] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0663] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0664] Finally, the PEG-TNF-alpha conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0665] In an alternative embodiment, Method 3 is carried out as follows.
TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). TNF-alpha is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0666] Finally, the PEG-TNF-alpha conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected and then
subjected to UF/DF. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

Method 4:

[0667] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of TNF-alpha is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg TNF-alpha/ml.
Subsequently, an 5 mM aqueous sodium periodate solution is added within
15 minutes to give a final concentration of 100 μM, followed by
addition of an 50 mM aqueous m-toluidine solution to get a final
concentration of 10 mM within a time period of 30 minutes. Then the
aminooxy-PEG reagent with a MW of 20 kD (described above) is added to
give a 20-fold molar reagent excess. After correction of the pH to 6.0
the mixture is incubated for 2 h in the dark at room temperature under
gentle stirring and quenched for 15 min at room temperature by the
addition of an 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.

[0668] The PEG-TNF-alpha conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0669] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 39

PEGylation of Insulin Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0670] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art. Insulin is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Insulin is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 7.5 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0671] The retentate containing oxidized insulin is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0672] Finally, the PEG-insulin conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0673] In an alternative embodiment, Method 1 is carried out as follows.
As described herein, the amino acid sequence of insulin is first modified
to incorporate at least one glycosylation site. Following purification,
insulin is glycosylated in vitro according to methods known in the art.
Insulin is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0674] The retentate containing oxidized insulin is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0675] Finally, the PEG-insulin conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0676] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0677] Insulin is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 200 μM. The oxidation reaction
is carried out for 30+/-5 min at a temperature (T) of T=+22+/-2°
C. Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0678] The oxidized insulin is further purified by ion exchange
chromatography. The oxidized insulin containing fractions of the eluate
are collected and used for the conjugation reaction.

[0679] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
insulin within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking.

[0680] The obtained PEG-insulin conjugate is further purified by ion
exchange chromatography. The PEG-insulin conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

[0681] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0682] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0683] Insulin is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0684] Finally, the PEG-insulin conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0685] In an alternative embodiment, Method 3 is carried out as follows.
As described herein, the amino acid sequence of insulin is first modified
to incorporate at least one glycosylation site. Following purification,
insulin is glycosylated in vitro according to methods known in the art.
Insulin is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Insulin is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0686] Finally, the insulin-conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected and then
subjected to UF/DF. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

Method 4:

[0687] As described herein, the amino acid sequence of insulin is first
modified to incorporate at least one glycosylation site. Following
purification, insulin is glycosylated in vitro according to methods known
in the art.

[0688] Insulin is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of insulin is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg insulin/ml.
Subsequently an 5 mM aqueous sodium periodate solution is added within 15
minutes to give a final concentration of 100 μM, followed by addition
of an 50 mM aqueous m-toluidine solution to get a final concentration of
10 mM within a time period of 30 minutes. Then the aminooxy-PEG reagent
with a MW of 20 kD (described above) is added to give a 20-fold molar
reagent excess. After correction of the pH to 6.0 the mixture is
incubated for 2 h in the dark at room temperature under gentle stirring
and quenched for 15 min at room temperature by the addition of a 1 M
aqueous L-cysteine solution to give a final concentration of 10 mM.

[0689] The PEG-insulin conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0690] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 40

PEGylation of Interferon-alpha Using an aminooxy-PEG Reagent and
m-toluidine as a Nucleophilic Catalyst

Method 1:

[0691] Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 7.5 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0692] The retentate containing oxidized interferon-alpha is next mixed
with an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added
to give a 5-fold molar reagent excess. This mixture is incubated for 2.5
h at room temperature in the dark under gentle stirring.

[0693] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example, 1.5
mg protein/ml gel is loaded on the column equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with 50 mM
Hepes buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0694] In an alternative embodiment, Method 1 is carried out as follows.
Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 7.5 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0695] The retentate containing oxidized interferon-alpha is next mixed
with an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added
to give a 5-fold molar reagent excess. This mixture is incubated for 2.5
h at room temperature in the dark under gentle stirring.

[0696] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography The conjugate containing fractions are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0697] Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-alpha is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 μM. The
oxidation reaction is carried out for 30+/-5 min at a temperature (T) of
+22+/-2° C. Then the reaction is stopped by addition of an aqueous
L-cysteine solution (1 M) within 15 minutes at T=+22+/-2° C. to
give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0698] The oxidized interferon-alpha is further purified by ion exchange
chromatography. The oxidized interferon-alpha containing fractions of the
eluate are collected and used for the conjugation reaction.

[0699] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
interferon-alpha within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0700] The obtained PEG-interferon-alpha conjugate is further purified by
ion exchange chromatography. The PEG-interferon alpha conjugate
containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off (Millipore).

[0701] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0702] Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous sodium
periodate solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0703] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is
loaded on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using a membrane. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.

[0704] In an alternative embodiment, Method 3 is carried out as follows.
Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous sodium
periodate solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0705] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions are
collected and then subjected to UF/DF using a membrane. The preparation
is analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4;

[0706] Interferon-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of interferon-alpha is transferred or
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of 2 mg
interferon-alpha/ml. Subsequently, an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration of 100
μM, followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30 minutes.
Then the aminooxy-PEG reagent with a MW of 20 kD (described above) is
added to give a 20-fold molar reagent excess. After correction of the pH
to 6.0 the mixture is incubated for 2 h in the dark at room temperature
under gentle stirring and quenched for 15 min at room temperature by the
addition of an 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.

[0707] The PEG-interferon-alpha conjugate is purified by means of ion
exchange chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes, 5 mM
CaCl2, pH 7.5).

[0708] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 41

PEGylation of Interferon-gamma Using an aminooxy-PEG Reagent and
m-toluidine as a Nucleophilic Catalyst

Method 1:

[0709] Interferon-gamma is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg
Interferon-gamma is dissolved in 5 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl). 100 μl of an aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated for 1
h in the dark at 4° C. under gentle stirring and quenched for 15
min at room temperature by the addition of 50 μl of a 1 M aqueous
cysteine solution. The mixture is subsequently subjected to UF/DF
employing Vivaspin 15R 10 kD centrifugal filtrators to remove excess
periodate, quencher and the byproducts thereof.

[0710] The retentate (approx. 7 ml), containing oxidized interferon-gamma,
is mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PEG reagent with
a MW of 20 kD (described above) is added to give a 5-fold molar reagent
excess. This mixture is incubated for 2.5 h at RT in the dark under
gentle stirring.

[0711] Finally, the PEG-interferon-gamma conjugate is purified by
ion-exchange chromatography on SP Sepharose FF. The reaction mixture is
diluted with 20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml
HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with Buffer B
(50 mM Hepes, 1 M NaCl, pH 6.5). Free interferon-gamma is eluted by
washing the column with 25% Buffer B and the conjugate at 50% Buffer B.
The conjugate containing fractions are concentrated by UF/DF using a 10
kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
histidine buffer, pH 6.9 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art. For the
PEG-interferon-gamma conjugate a specific activity of >50% in
comparison to native Interferon gamma is determined. The conjugate is
additionally analytically characterized by Size Exclusion HPLC using a
Agilent 1200 HPLC system equipped with a Shodex KW 803 column under
conditions as previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
Interferon-gamma.

Method 2:

[0712] Interferon-gamma is PEGylated by use of a linear 2010 PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan).
Interferon-gamma is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to get a
final protein concentration of 1.0+/-0.25 mg/ml. Then the pH of the
solution is corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 μM. The oxidation
reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2° C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at T=+22+/-2°
C. to give a final concentration of 10 mM in the reaction mixture and
incubation for 60+/-5 min.

[0713] The oxidized interferon-gamma is further purified by ion exchange
chromatography. The oxidized interferon-gamma containing fractions of the
eluate are collected and used for the conjugation reaction.

[0714] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
interferon-gamma within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. The reaction
mixture is incubated for 120+/-10 min. in the dark at a temperature (T)
of T=+22+/-2° C. under gentle shaking.

[0715] The obtained PEG-interferon-gamma conjugate is further purified by
ion exchange chromatography. The PEG-interferon-gamma conjugate
containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off (Millipore).

[0716] The conjugate prepared by use of this procedure are analytically
characterized by measuring total protein and biological activity
according to methods known in the art.

Method 3:

[0717] Interferon-gamma is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg
interferon-gamma is dissolved in ˜8 ml histidine-buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl). 200 μl of an aqueous sodium periodate
solution (5 mM) and 2 ml of an aqueous m-toluidine solution (50 mM) are
then added. Subsequently the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. The
mixture is incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the addition of
100 μl of 1 M aqueous cysteine solution.

[0718] Finally the PEG-interferon-gamma conjugate is purified by
ion-exchange chromatography on SP-Sepharose FF. The reaction mixture is
diluted with 20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml
HiPrep SP FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with Buffer B
(50 mM Hepes, 1 M NaCl, pH 6.5). Free interferon-gamma is eluted by
washing the column with 25% Buffer B and the conjugate at 50% Buffer B.
The conjugate containing fractions are concentrated by UF/DF using a 10
kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
histidine buffer, pH 6.9 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art. For the
PEG-interferon-gamma conjugate a specific activity of >50% in
comparison to native interferon-gamma is determined. The conjugate is
additionally analytically characterized by Size Exclusion HPLC using a
Agilent 1200 HPLC system equipped with a Shodex KW 803 column under
conditions as previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
interferon-gamma.

Method 4:

[0719] Interferon-gamma is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of reagent
is the Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of interferon-gamma is transferred or
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of 2 mg
interferon-gamma/ml. Subsequently an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration of 100
μM, followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30 minutes.
Then the aminooxy-PEG reagent with a MW of 20 kD (described above) is
added to give a 20-fold molar reagent excess. After correction of the pH
to 6.0 the mixture is incubated for 2 h in the dark at room temperature
under gentle stirring and quenched for 15 min at room temperature by the
addition of an 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.

[0720] The PEG-interferon-gamma conjugate is purified by means of ion
exchange chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes, 5 mM
CaCl2, pH 7.5).

[0721] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 42

PEGylation of G-CSF Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0722] G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0723] The retentate containing oxidized G-CSF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0724] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0725] In an alternative embodiment, Method 1 is carried out as follows.
G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0726] The retentate containing oxidized G-CSF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0727] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0728] G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently a 40 mM aqueous sodium periodate solution is added within 10
minutes to give a concentration of 200 μM. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of T=+22+/-2° C.
Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0729] The oxidized G-CSF is further purified by ion exchange
chromatography. The oxidized G-CSF containing fractions of the eluate are
collected and used for the conjugation reaction.

[0730] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized G-CSF
within a maximum time period (t) of 15 minutes under gentle stirring.
Then an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. The reaction mixture is incubated
for 120+/-10 min. in the dark at a temperature (T) of T=+22+/-2°
C. under gentle shaking.

[0731] The obtained PEG-G-CSF conjugate is further purified by ion
exchange chromatography. The PEG-G-CSF conjugate containing fractions are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

Method 3:

[0732] G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0733] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0734] In an alternative embodiment, Method 3 is carried out as follows.
G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent containing
an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). G-CSF is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0735] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using a membrane. The preparation
is analytically characterized by measuring total protein (Bradford) and
biological activity according to methods known in the art.

Method 4:

[0736] G-CSF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of G-CSF is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg G-CSF/ml. Subsequently,
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0737] The G-CSF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0738] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 43

PEGylation of Humira Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0739] Humira is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0740] The retentate containing oxidized Humira is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0741] Finally, the PEG-Humira conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0742] In an alternative embodiment, Method 1 is carried out as follows.
Humira is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0743] The retentate containing oxidized Humira is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0744] Finally, the PEG-Humira conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW cutoff
membrane. The preparation is next analytically characterized by measuring
total protein (Coomassie, Bradford) and biological activity according to
methods known in the art.

Method 2:

[0745] Humira is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently a 40 mM aqueous sodium periodate solution is added within 10
minutes to give a concentration of 200 μM. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of T=+22+/-2° C.
Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0746] The oxidized Humira is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the eluate
are collected and used for the conjugation reaction.

[0747] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
Humira within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking.

[0748] The obtained PEG-Humira conjugate is further purified by ion
exchange chromatography. The PEG-Humira conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

Method 3:

[0749] Humira is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently, the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0750] Finally, the PEG-Humira conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0751] In an alternative embodiment, Method 3 is carried out as follows.
Humira is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Humira is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0752] Finally, the PEG-Humira conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected and then
subjected to UF/DF using a membrane. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.

Method 4:

[0753] Humira is PEGylated by use of a linear 2010 PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of HJumira is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg Humira/ml. Subsequently
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of a 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0754] The Humira conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0755] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 44

PEGylation of Prolia Using an aminooxy-PEG Reagent and m-toluidine as a
Nucleophilic Catalyst

Method 1:

[0756] Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Prolia is
dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl, 5 mM CaCl2). An aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate, quencher and
the byproducts thereof.

[0757] The retentate containing oxidized Prolia is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at room
temperature. Aminooxy-PEG reagent with a MW of 20 kD is then added to
give a 5-fold molar reagent excess. This mixture is incubated for 2.5 h
at room temperature in the dark under gentle stirring.

[0758] Finally, the PEG-Prolia conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes buffer
containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and is then
subjected to UF/DF using an appropriate MW cutoff membrane. The
preparation is next analytically characterized by measuring total protein
(Coomassie, Bradford) and biological activity according to methods known
in the art.

[0759] In an alternative embodiment, Method 1 is carried out as follows.
Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg rFIX
is dissolved in 5 ml histidine-buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl). 100 μl of an aqueous sodium periodate solution (5 mM) is then
added and the reaction mixture is incubated for 1 h in the dark at
4° C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 50 μl of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin 15R 10 kD centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.

[0760] The retentate (approx. 7 ml), containing oxidized Prolia, is mixed
with 2 ml of an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Then aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at RT in the dark under gentle stirring.

[0761] Finally the PEG-Prolia conjugate is purified by ion-exchange
chromatography on SP Sepharose FF. The reaction mixture is diluted with
20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SP FF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with
Buffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 M NaCl,
pH 6.5). Free Prolia is eluted by washing the column with 25% Buffer B
and the conjugate at 50% Buffer B. The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against histidine buffer, pH 6.9 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.
For the PEG-Prolia conjugate a specific activity of >50% in comparison
to native Prolia is determined. The conjugate is additionally
analytically characterized by Size Exclusion HPLC using a Agilent 1200
HPLC system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006; 46:1959-77). It
is shown that the preparation contains no free Prolia.

Method 2:

[0762] Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). Prolia is
transferred or dissolved in reaction buffer (e.g. 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added within
10 minutes to give a concentration of 200 μM. The oxidation reaction
is carried out for 30+/-5 min at a temperature (T) of T=+22+/-2°
C. Then the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2° C. to give a final
concentration of 10 mM in the reaction mixture and incubation for 60+/-5
min.

[0763] The oxidized Prolia is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the eluate
are collected and used for the conjugation reaction.

[0764] The aminooxy-PEG reagent with a MW of 20 kD reagent is added in a
50-fold molar excess to the eluate containing the purified oxidized
Prolia within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added within 15
minutes to get a final concentration of 10 mM. The reaction mixture is
incubated for 120+/-10 min. in the dark at a temperature (T) of
T=+22+/-2° C. under gentle shaking.

[0765] The obtained PEG-Prolia conjugate is further purified by ion
exchange chromatography. The PEG-Prolia conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose with an appropriate molecular
weight cut off (Millipore).

Method 3:

[0766] Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). EPO is
dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at room
temperature under gentle stirring and quenched for 15 min at room
temperature by the addition of 8 μl of aqueous cysteine solution (1
M).

[0767] Finally, the PEG-Prolia conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded on the
column pre equilibrated with 50 mM Hepes buffer, pH 7.4 containing 5 mM
CaCl2. The conjugate is eluted with 50 mM Hepes buffer containing 5 mM
CaCl2 and 500 mM sodium chloride, pH 7.4 and is then subjected to UF/DF
using a membrane. The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity according to
methods known in the art.

[0768] In an alternative embodiment, Method 3 is carried out as follows.

[0769] Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). 10 mg Prolia
is dissolved in ˜8 ml histidine buffer, pH 6.0 (20 mM L-histidine,
150 mM NaCl). 200 μl of an aqueous sodium periodate solution (5 mM)
and 2 ml of an aqueous m-toluidine solution (50 mM) are then added.
Subsequently, the aminooxy-PEG reagent with a MW of 20 kD (described
above) is added to give a 5-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle stirring
and quenched for 15 min at room temperature by the addition of 100 μl
of 1 M aqueous cysteine solution.

[0770] Finally the PEG-Prolia conjugate is purified by ion-exchange
chromatography on SP-Sepharose FF. The reaction mixture is diluted with
20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated with
Buffer A. Then the column is eluted with Buffer B (50 mM Hepes, 1 M NaCl,
pH 6.5). Free Prolia is eluted by washing the column with 25% Buffer B
and the conjugate at 50% Buffer B. The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against histidine buffer, pH 6.9 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total protein
(Bradford) and biological activity according to methods known in the art.
For the PEG-Prolia conjugate a specific activity of >50% in comparison
to native Prolia is determined. The conjugate is additionally
analytically characterized by Size Exclusion HPLC using a Agilent 1200
HPLC system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006; 46:1959-77). It
is shown that the preparation contains no free Prolia.

Method 4:

[0771] Prolia is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is the
Sunbright® CA series from NOF (NOF Corp., Tokyo, Japan). An initial
concentration or weight of HJumira is transferred or dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) to get a final protein concentration of 2 mg Prolia/ml. Subsequently
an 5 mM aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 μM, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM within
a time period of 30 minutes. Then the aminooxy-PEG reagent with a MW of
20 kD (described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of an 1 M aqueous L-cysteine solution to
give a final concentration of 10 mM.

[0772] The Prolia conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the eluate
are concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final diafiltration step
is performed against Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).

[0773] The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and biological activity according to
known methods.

Example 45

PEGylation of a Therapeutic Protein Using Branched PEG

[0774] PEGylation of a therapeutic protein of the invention may be
extended to a branched or linear PEGylation reagent, which is made of an
aldehyde and a suitable linker containing an active aminooxy group.